Combination of Anti-CD20 Antibody and PI3 Kinase Selective Inhibitor

ABSTRACT

Highly effective combinations of a compound of formula A (a PI3Kδ selective inhibitor) and anti-CD20 antibodies are provided herein for the treatment and amelioration of PI3Kδ and/or CD20 mediated diseases and disorders. In particular, the combination can be used to treat cancers and autoimmune diseases. More particularly, the invention provides for a combination of a compound of formula A, or stereoisomers thereof, and ublituximab for the treatment and/or amerioration of hematological malignancies such as leukemia and lymphoma. The invention also provides for a combination of a compound of formula A, or stereoisomers thereof, and anti-CD20 antibodies, including ublituximab for the treatment and/or amelioration of multiple scelrosis.

This application is a continuation of U.S. Serial No. U.S. Ser. No.15/635,733, filed Jun. 28, 2017, which is a continuation of U.S. SerialNo. U.S. Ser. No. 14/440,139, filed May 1, 2015, now U.S. Pat. No.9,694,071, issued Jul. 4, 2017, which is a national stage filing under35 U.S. § 371 of Intl. Appln. No. PCT/US2013/067956, filed Nov. 1, 2013,which claims priority under 35 U.S.C. § 119(e) of provisionalapplication U.S. Ser. No. 61/771,812, filed Mar. 2, 2013, and whichclaims priority of Indian Appln. No. 4595/CHE/2012, filed Nov. 2, 2012,each of which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, identified by the name43-101us3_ST25.txt, created Oct. 30, 2013 with a size of 8000 bytes andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

FIELD OF THE INVENTION

Highly effective combinations of a compound of formula A (a PI3Kδselective inhibitor) and anti-CD20 antibodies are provided herein forthe treatment and amelioration of PI3Kδ and/or CD20 mediated diseasesand disorderes. In particular, the combination can be used to treatcancers and autoimmune diseases. More particularly, the inventionprovided for a combination of a compound of formula A, or stereoisomersthereof, and ublituximab for the treatment and/or amerioration ofhematological malignancies such as leukemia and lymphoma.

BACKGROUND OF THE INVENTION

There is considerable evidence indicating that both PI3Kδ enzymes andCD20 contribute individually to tumourigenesis in a wide variety ofhuman cancers and especially in hematological malignancies. Thephosphoinositide 3-kinases (PI3Ks) are a family of enzymes that regulatediverse biological functions in every cell type by generatingphosphoinositide second-messenger molecules. As the activity of thesephosphoinositide second messengers is determined by theirphosphorylation state, the kinases and phosphatises that act to modifythese lipids are central to the correct execution of intracellularsignaling events. PI3Ks phosphorylate lipids at the 3-hydroxyl residueof an inositol ring (Whitman et al., Nature 332:664 (1988)) to generatephosphorylated phospholipids (PIP3s) which act as second messengersrecruiting kinases with lipid binding domains (including plekstrinhomology (PH) regions), such as Akt and phosphoinositide-dependentkinase-1 (PDK1). Binding of Akt to membrane PIP3s causes thetranslocation of Akt to the plasma membrane, bringing Akt into contactwith PDK1, which is responsible for activating Akt. The tumor-suppressorphosphatase, PTEN (Phosphatase and Tensin homolog deleted on chromosomeTen), dephosphorylates PIP3 and therefore acts as a negative regulatorof Akt activation. The PI3-kinases Akt and PDK1 are important in theregulation of many cellular processes including cell cycle regulation,proliferation, survival, apoptosis and motility and are significantcomponents of the molecular mechanisms of diseases such as cancer,diabetes and immune inflammation (Vivanco et al., Nature Rev. Cancer2:489 (2002); Phillips et al., Cancer 83:41 (1998)).

The PI3Ks family is constituted by four different classes: Classes I,II, III and IV. Classes I-III are lipid kinases and Class IV areserine/threonine protein kinases.

The members of the Class I family of PI3Ks are dimers of a regulatoryand a catalytic subunit. The Class I family consists of four isoforms,determined by the 110 kDa catalytic subunits α, β, γ and δ. See EngelmanJ. A., Nat Rev Genet 7:606-619 (2006); Carnero A., Curr Cancer DrugTargets 8:187-198 (2008); and Vanhaesebroeck B., Trends Biochem Sci30:194-204 (2005). Class I can be subdivided into two subclasses: ClassIa, formed by the combination of pl10 α, β, and δ, and a regulatorysubunit (p85, p55 or p50); and Class Ib, formed by p110 γ and p101regulatory subunits.

Studies regarding PI3K and related protein kinase pathways have beenpublished by various research groups, including, Liu et al., NatureReviews Drug Discovery 8:627-644 (2009); Nathan et al, Mol. Cancer Ther.8(1) (2009); and Marone et al., Biochimica et Biophysica Acta1784:159-185 (2008). Two known PI3K inhibitors, LY294002 and Wortmannin,are non-specific PI3K inhibitors as they do not distinguish the fourmembers of Class I PI3K: α, β, γ, and δ. A number of PI3K inhibitorshave entered clinical trials for the treatment of cancers, and varioustypes of cancers, including breast cancer, non-small cell lung cancer(NSCLC), and hematological cancers, are being considered as areas oftherapeutic interest.

CD20 is a hydrophobic transmembrane protein with a molecular weight of35-37 kDa which is present on the surface of mature B lymphocytes. It isexpressed during the development of B lymphocyte cells (B cells) as fromthe early pre-B stage until differentiation into plasmocytes, a stage atwhich this expression disappears. CD20 is present on both normal Blymphocytes and malignant B cells including most non-Hodgkin's B-celllymphomas (NHL) and B-type Chronic Lymphocytic Leukemia's (B-CLL). TheCD20 antigen is not expressed on haematopoietic stem cells or onplasmocytes.

Anti-CD20 antibodies have been, and continue to be, developed for thetreatment of B-cell diseases. Successes have been reported for theanti-CD20 antibody rituximab. However, there are a substantial number ofpatients who are refractory to treatment with rituximab or who developresistance in the course of prolonged treatment with rituximab (used asa single agent or even in combination with chemotherapeutic regimens).

Accordingly, there is a need for more effective therapies for thetreatment and/or amelioration of diseases or disorders associated withmodulation of PI3Kδ enzymes and/or CD20 protein, and in particular forthe treatment and or amelioration of B-cell diseases.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a combination comprising of acompound of formula (A) that is a PI3Kδ selective inhibitor,

and a stereoisomer thereof, a tautomer thereof, pharmaceuticallyacceptable salts, solvates, and prodrugs thereof, and at least oneanti-CD20 antibody.

The combination is suitable for use in the treatment of a PI3Kδ enzyme-and/or CD20 protein-associated disease, disorder or condition, e.g., aproliferative disease such as cancer. In particular, the combination issuitable for the treatment and or amelioration of B-cell diseases, e.g.,hematological malignancies.

Thus, in some embodiments, methods of inhibiting proliferation of a cellpopulation are provided. In some embodiments, the method comprisescontacting the population with a combination comprising (i) a compoundof formula A, a stereoisomer thereof, a tautomer thereof, or apharmaceutically acceptable salt, solvate, or prodrug thereof, (ii) andan anti-CD20 antibody or antigen-binding fragment thereof, wherein theanti-CD20 antibody or fragment binds to the same epitope as ublituximab.

In some embodiments, the method comprises contacting the cell populationwith a combination comprising (i) a compound of formula A, astereoisomer thereof, a tautomer thereof, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, and (ii) an anti-CD20antibody or antigen-binding fragment thereof, wherein the anti-CD20antibody or fragment exhibits a high affinity to Fc-gammaRIII (CD16).

In some embodiments, the method comprises contacting the cell populationwith a combination comprising (i) a compound of formula A, astereoisomer thereof, a tautomer thereof, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, and (ii) an anti-CD20antibody or antigen-binding fragment thereof, wherein the fucose contentof the antibody or fragment is less than 65%.

In some embodiments, the method comprises contacting the cell populationwith a combination comprising (i) a compound of formula A, astereoisomer thereof, a tautomer thereof, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof, and (ii) an anti-CD20antibody or antigen-binding fragment thereof, wherein the antibody orfragment comprises the VH CDR1, CDR2 and CDR3 region of sequences SEQ IDNO:1, 2, and 3, and the VL CDR1, CDR2 and CDR3 region of sequences SEQID NO:6, 7, and 8. In some embodiments, the anti-CD20 antibody orantigen-binding fragment thereof comprises the VH of SEQ ID NO:4 and theVL of SEQ ID NO:9. In some embodiments, the anti-CD20 antibody isublituximab.

In some embodiments, the compound of fomula A is

-   (RS)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    or-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one.

In some embodiments, the compound of fomula A is(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one.

In some embodiments, a method of inhibiting proliferation of a cellpopulation comprises contacting the population with a combinationcomprising (i) at least one compound selected from the group consistingof2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;and(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and (ii) at leastone anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, a method of inhibiting proliferation of a cellpopulation comprises contacting the population with a combinationcomprising (i) a compound selected from the group consisting of2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;and(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and (ii) at leastone anti-CD20 antibody or antigen-binding fragment thereof, wherein theanti-CD20 antibody or fragment thereof is selected from the groupconsisting of antibodies and fragments thereof that bind to the sameepitope as ublituximab, rituximab, ofatumumab, ocrelizumab, veltuzumab,GA101, AME-133v, PRO131921, tositumomab, hA20, and PR070769.

In some embodiments, the population is contacted with a compositioncomprising (i) a compound selected from the group consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and    (ii) the anti-CD20 antibody.

In some embodiments, the population is contacted with (i) a firstcomposition comprising a compound selected from the group consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,    and    (ii) a second composition comprising the anti-CD20 antibody.

In some embodiments, the population comprises B-cells.

In some embodiments, the population is in a human subject.

In some embodiments, the subject has a disease or disorder associatedwith excessive B-cell proliferation.

In some embodiments, the subject has cancer. In some embodiments, thecancer is a hematological malignancy. In some embodiments, thehematological malignancy is lymphoma or leukemia. In some embodiments,the hematological malignancy is selected from the group consisting ofacute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiplemyeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL),follicular lymphoma, Waldenstrom's macroglobulinemia (WM), B-celllymphoma and diffuse large B-cell lymphoma (DLBCL). In some embodiments,the cancer overexpresses CD20. In some embodiments, the cancer isrefractory to chemotherapy.

In some embodiments, the subject has an autoimmune disease or disorder.In some embodiments, the autoimmune disease or disorder is allergicrhinitis, asthma, chronic obstructive pulmonary disease (COPD), orrheumatoid arthritis.

In some embodiments, the subject is refractory to rituximab.

In some embodiments, the subject has previously been treated withchemotherapy, rituximab, or a combination thereof.

In some embodiments, the(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-oneand the anti-CD20 antibody or fragment are administered sequentially.

In some embodiments, the(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-oneand the anti-CD20 antibody or fragment are administered simultaneously.In some embodiments, the(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-oneand the anti-CD20 antibody or fragment are contained in the samepharmaceutical composition. In some embodiments, the(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,and the anti-CD20 antibody or fragment are in separate pharmaceuticalcompositions.

In some embodiments, the method further comprises administering at leastone additional therapeutic agent to the subject. In some embodiments,the at least one additional therapeutic agent is selected from the groupconsisting of a proteasome inhibitor, Bortezomib (Velcade©), Carfilzomib(PR-171), PR-047, disulfiram, lactacystin, PS-519, eponemycin,epoxomycin, aclacinomycin, CEP-1612, MG-132, CVT-63417, PS-341, vinylsulfone tripeptide inhibitors, ritonavir, PI-083,(+/−)-7-methylomuralide, (−)-7-methylomuralide, lenalidomide, andcombinations thereof.

In some embodiments, the method further comprises administering at leasttwo additional therapeutic agents to the subject, wherein the at leasttwo additional therapeutic agents are selected from the group consistingof: a) CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone); b)R-CHOP (rituximab-CHOP); c) hyperCV AD (hyperfractionatedcyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate,cytarabine); d) R-hyperCV AD (rituximab-hyperCV AD); e) FCM(fludarabine, cyclophosphamide, mitoxantrone); f) R-FCM (rituximab,fludarabine, cyclophosphamide, mitoxantrone); g) bortezomib andrituximab; h) temsirolimus and rituximab; i) temsirolimus and Velcade®;j) Iodine-131 tositumomab (Bexxar®) and CHOP; k) CVP (cyclophosphamide,vincristine, prednisone); 1) R-CVP (rituximab-CVP); m) ICE(iphosphamide, carboplatin, etoposide); n) R-ICE (rituximab-ICE); o) FCR(fludarabine, cyclophosphamide, rituximab); p) FR (fludarabine,rituximab); and q) D.T. PACE (Dexamethasone, Thalidomide, Cisplatin,Adriamycin, Cyclophosphamide, Etoposide).

In some embodiments, methods for depleting B-cells are provided. In someembodiments, the method comprises contacting a composition comprisingB-cells with (i) at least one compound of formula A selected from thegroup consisting of2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;and(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and (ii) at leastone anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, method for promoting apoptosis are provided. Insome embodiments, the methods comprise contacting a B-cell with (i) atleast one compound of formula A selected from the group consisting of2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;and(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and (ii) at leastone anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, methods for promoting cell-cycle arrest areprovided. In some embodiments, the methods comprise contacting a cellwith (i) at least one compound of formula A selected from the groupconsisting of2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;and(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and (ii) at leastone anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, the compound of formula A and the anti-CD20antibody or fragment are delivered sequentially.

In some embodiments, the compound of formula A and the anti-CD20antibody or fragment are delivered simultaneously. In some embodiments,the compound of formula A and the anti-CD20 antibody or fragment aredelivered in the same composition. In some embodiments, the compound offormula A and the anti-CD20 antibody or fragment are delivered inseparate compositions.

Kits are also provided herein. In some embodiments, the kit comprises(i) a compound of formula A, a stereoisomer thereof, a tautomer thereof,or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and(ii) instructions for using the compound in combination with ananti-CD20 antibody or antigen-binding fragment thereof, wherein theanti-CD20 antibody or fragment (a) binds to the same epitope asublituximab, (b) exhibits a high affinity to Fc-gammaRIII (CD16), or (c)or has a fucose content of less than 65%.

In some embodiments, the kit comprises (i) at least one compoundselected from the group consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and    (ii) instructions for using the compound in combination with an    anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, the kit further comprises the anti-CD20 antibody orfragment.

In some embodiments, the kit comprises (i) at least one anti-CD20antibody or antigen-binding fragment thereof and (ii) instructions forusing the anti-CD20 antibody or fragment in combination with at leastone compound selected from the group consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,    wherein the anti-CD20 antibody or a fragment thereof (a) binds to    the same epitope as ublituximab, (b) exhibits a high affinity to    Fc-gammaRIII (CD16), or (c) has a fucose of less than 65%.

In some embodiments, the kit comprises (i) at least one anti-CD20antibody or antigen-binding fragment thereof and (ii) instructions forusing the anti-CD20 antibody or fragment in combination with at leastone compound selected from the group consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and,-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one.

In some embodiments, the kit comprises (i) a compound selected from thegroup consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and    (ii) an anti-CD20 antibody or antigen-binding fragment thereof,    wherein the anti-CD20 antibody or fragment (a) binds to the same    epitope as ublituximab, (b) exhibits a high affinity to Fc-gammaRIII    (CD16), or (c) has a fucose content of less than 65%.

In some embodiments, the kit comprises (i) a compound selected from thegroup consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and    (ii) an anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, the anti-CD20 antibody or fragment and the compoundare contained within the same composition.

In some embodiments, the anti-CD20 antibody or fragment and the compoundare in separate compositions.

In some embodiments, the kit further comprises one or more additionalactive agents.

Pharmaceutical compositions are also provided herein. In someembodiments, the pharmaceutical composition comprises (i) a compoundselected from the group consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and    (ii) an anti-CD20 antibody or antigen-binding fragment thereof.

In some embodiments, the pharmaceutical composition comprises (i) acompound selected from the group consisting of

-   2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;-   (S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one;    and-   (R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)    ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one, and    (ii) an anti-CD20 antibody or antigen-binding fragment thereof,    wherein the anti-CD20 antibody or fragment (a) binds to the same    epitope as ublituximab, (b) exhibits a high affinity to Fc-gammaRIII    (CD16), or (c) has a fucose content less than 65%.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Bar graphs showing the effect of S-isomer of a compound offormula A and Ublituximab on CD19-positive cell depletion (left) andCD20-positive cell depletion (right) from human whole blood.

FIG. 2: Bar graphs showing the effect of S-isomer of a compound offormula A and Ublituximab on LPS-induced CD19-positive cellproliferation.

FIG. 3: Bar graphs showing the effect of S-isomer of a compound offormula A and Ublituximab on LPS-induced CD20-positive cellproliferation.

FIG. 4: Bar graphs showing the effect of S-isomer of a compound offormula A and Ublituximab on apoptosis in Daudi, RPMI-8226, Raji, andU266B1 cells.

FIG. 5: Histograms showing the effect of S-isomer of a compound offormula A on cell cycle in U226B1 cells.

FIG. 6: Histograms showing the effect of the anti-CD20 antibodyublituximab on cell cycle in U226B1 cells.

FIG. 7: Histograms showing the effect of S-isomer of a compound offormula A and the anti-CD20 antibody ublituximab on cell cycle in U226B1cells.

FIG. 8: Histograms showing the effect of S-isomer of a compound offormula A on cell cycle in Raji cells.

FIG. 9: Histograms showing the effect of the anti-CD20 antibodyublituximab on cell cycle in Raji cells.

FIG. 10: Histograms showing the effect of S-isomer of a compound offormula A and the anti-CD20 antibody ublituximab on cell cycle in Rajicells.

FIG. 11: Plots of caspase 3 activity showing the effect of S-isomer of acompound of formula A and Ublituximab on apoptosis in LY1 cells.

FIG. 12: Plots of caspase 3 activity showing the effect of S-isomer of acompound of formula A and Ublituximab on apoptosis in Raji cells.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term “CD20” (also known as B lymphocyte CD20 antigen, MS4A1, Blymphocyte surface antigen B1, Bp35, Leukocyte surface antigen Leu-16)refers to any native CD20, unless otherwise indicated. The term “CD20”encompasses “full-length,” unprocessed CD20 as well as any form of CD20that results from processing within the cell. The term also encompassesnaturally occurring variants of CD20, e.g., splice variants, allelicvariants and isoforms. The CD20 polypeptides described herein can beisolated from a variety of sources, such as from human tissue types orfrom another source, or prepared by recombinant or synthetic methods.Examples of CD20 sequences include, but are not limited to NCBIreference numbers NP_068769.2 and NP_690605.1.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any of the five major classesof immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses(isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), basedon the identity of their heavy-chain constant domains referred to asalpha, delta, epsilon, gamma, and mu, respectively. The differentclasses of immunoglobulins have different and well known subunitstructures and three-dimensional configurations. Antibodies can be nakedor conjugated to other molecules such as toxins, radioisotopes,proteins, etc.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such as CD20. Ina certain embodiment, blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen. Desirably, the biological activity is reduced by 10%, 20%, 30%,50%, 70%, 80%, 90%, 95%, or even 100%.

The term “anti-CD20 antibody” or “an antibody that binds to CD20” refersto an antibody that is capable of binding CD20 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD20. The extent of binding of an anti-CD20 antibodyto an unrelated, non-CD20 protein is less than about 10% of the bindingof the antibody to CD20 as measured, e.g., by a radioimmunoassay (RIA).In certain embodiments, an antibody that binds to CD20 has adissociation constant (Kd) of ≤1 M, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g., mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,Science, 239:1534-1536 (1988)). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539 or5,639,641.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al J. Molec. Biol.273:927-948 (1997)). In addition, combinations of these two approachesare sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues can be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Chothiarefers instead to the location of the structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loopwhen numbered using the Kabat numbering convention varies between H32and H34 depending on the length of the loop (this is because the Kabatnumbering scheme places the insertions at H35A and H35B; if neither 35Anor 35B is present, the loop ends at 32; if only 35A is present, theloop ends at 33; if both 35A and 35B are present, the loop ends at 34).The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-HS8 H52-H156 H3 H95-H102 H95-H102 H95-H102

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedherein.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical Kd value. For example, an antibody which has an affinity foran antigen of “0.6 nM or better,” the antibody's affinity for theantigen is <0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM etc. or any valueless than 0.6 nM.

The phrase “substantially similar,” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicsmeasured by said values (e.g., Kd values). The difference between saidtwo values is less than about 50%, less than about 40%, less than about30%, less than about 20%, or less than about 10% as a function of thevalue for the reference/comparator antibody.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, an antibody, polynucleotide, vector, cell, or compositionwhich is isolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancers.

“Tumor” and “neoplasm” refer to any mass of tissue that result fromexcessive cell growth or proliferation, either benign (noncancerous) ormalignant (cancerous) including pre-cancerous lesions.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

A cell “population” can refer to a single cell or to multiple cells. Thecell or cells can be cells in culture or cells in an organism. Forexample, a cell population can be in a subject or patient.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulation can be sterile.

An “effective amount” of an antibody as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug can reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and in a certainembodiment, stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and in a certain embodiment, stop)tumor metastasis; inhibit, to some extent, tumor growth; and/or relieveto some extent one or more of the symptoms associated with the cancer.See the definition herein of “treating.” To the extent the drug canprevent growth and/or kill existing cancer cells, it can be cytostaticand/or cytotoxic. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically but not necessarily,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: reduction in cachexia, increase in survival time,elongation in time to tumor progression, reduction in tumor mass,reduction in tumor burden and/or a prolongation in time to tumormetastasis, time to tumor recurrence, tumor response, complete response,partial response, stable disease, progressive disease, progression freesurvival (PFS), overall survival (OS), each as measured by standards setby the National Cancer Institute and the U.S. Food and DrugAdministration for the approval of new drugs. See Johnson et al, J. Cin.Oncol. 21(7):1404-1411 (2003).

A “combination” of an anti-CD20 antibody and a PI3Kδ selective inhibitorrefers to an anti-CD20 antibody or fragment thereof and Compound A asdefined herein that are intended to be administered to the samepopulation of cells or to the same subject simultaneously, sequentially,or both simulteously and sequentially. Thus, by way of example,administration of an anti-CD20 antibody or fragment thereof preceedingor following (e.g., by an hour, day, week, or month) administration ofCompound A constitutes administration of a combination of an anti-CD20antibody or fragment thereof and Compound A. In addition, simultaneousadministration of an anti-CD20 antibody or fragment thereof and CompoundA also constitutes administration of a combination of the anti-CD20antibody or fragment thereof and Compound A, regardless of whether theanti-CD20 antibody or fragment thereof and Compound A are administeredtogether in a single pharmaceutical formulation or are administeredsimultaneously in separate pharmaceutical formulations by either thesame or different routes of administration.

A tumor which “does not respond,” “responds poorly,” or is “refractory”to treatment with an anti-CD20 antibody does not show statisticallysignificant improvement in response to an anti-CD20 antibody treatmentwhen compared to no treatment or treatment with placebo in a recognizedanimal model or human clinical trial, or which responds to an initialtreatment with anti-CD20 antibodies but grows as treatment continues.

The ability of a tumor or cell type to respond to an anti-CD20 antibodycan be tested using laboratory cell lines such as Raji or Wil2-S, orpatient donor cell lines. In addition, activity can be measured usingB-cell depletion assays, e.g., in whole blood from patients.

“Polynucleotide” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure canbe imparted before or after assembly of the polymer. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps,” substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars can be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, orcan be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls can also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages can be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

The term “vector” means a construct, which is capable of delivering, andexpressing, one or more gene(s) or sequence(s) of interest in a hostcell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, Proc. Nat. Acad. Sci., 87:2264-2268 (1990), as modified inKarlin et al., Proc. Nat. Acad. Sci., 90:5873-5877 (1993), andincorporated into the NBLAST and XBLAST programs (Altschul et al.,Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, GappedBLAST can be used as described in Altschul et al., Nucleic Acids Res.,25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods inEnzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) can be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.In certain embodiments, identity exists over a region of the sequencesthat is at least about 10, about 20, about 40-60 residues in length orany integral value therebetween, or over a longer region than 60-80residues, at least about 90-100 residues, or the sequences aresubstantially identical over the full length of the sequences beingcompared, such as the coding region of a nucleotide sequence forexample.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the FOLR1 to which the polypeptide or antibodybinds. Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen binding are well-known inthe art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993);Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al.Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

All numbers in this disclosure indicating amounts, ratios of materials,physical properties of materials, and/or use are to be understood asmodified by the word “about,” except as otherwise explicitly indicated.The term “about” when referring to a number or a numerical range meansthat the number or numerical range referred to is an approximationwithin experimental variability (or within statistical experimentalerror), and thus the number or numerical range can vary from, forexample, between 1% and 15% of the stated number or numerical range.

The compound of the invention can contain one or more asymmetric centers(chiral centers) and can thus give rise to enantiomers, diastereomers,and other stereoisomeric forms that can be defined, in terms of absolutestereochemistry, as (R)- or (S)-. The present disclosure is meant toencompass all such possible forms, as well as their racemic and resolvedforms and mixtures thereof. The individual enantiomers can be separatedaccording to methods known in the art in view of the present disclosure.

As used herein, the term “stereoisomers” is a general term for allisomers of individual molecules that differ only in the orientation oftheir atoms in space. It includes enantiomers and isomers of compoundswith more than one chiral center that are not mirror images of oneanother (diastereomers).

The term “chiral center” refers to a carbon atom to which four differentgroups are attached.

The terms “enantiomer” and “enantiomeric” refer to a molecule thatcannot be superimposed on its mirror image and hence is optically activewherein the enantiomer rotates the plane of polarized light in onedirection and its mirror image compound rotates the plane of polarizedlight in the opposite direction.

The term “racemic” refers to a mixture of equal parts of enantiomers andwhich mixture is optically inactive.

The term “resolution” refers to the separation, concentration ordepletion of one of the two enantiomeric forms of a molecule.

The present disclosure encompasses solvates of compounds of theinvention. Solvates typically do not significantly alter thephysiological activity or toxicity of the compounds, and as such mayfunction as pharmacological equivalents. The term “solvate” as usedherein is a combination, physical association and/or solvation of acompound of the present disclosure with a solvent molecule, e.g. adisolvate, monosolvate or hemisolvate, where the ratio of solventmolecule to compound of the present disclosure is about 2:1, about 1:1or about 1:2, respectively. This physical association involves varyingdegrees of ionic and covalent bonding, including hydrogen bonding. Incertain instances, the solvate can be isolated, such as when one or moresolvent molecules are incorporated into the crystal lattice of acrystalline solid. Thus, “solvate” encompasses both solution-phase andisolatable solvates. Compounds of the invention can be present assolvated forms with a pharmaceutically acceptable solvent, such aswater, methanol, ethanol, and the like, and it is intended that thedisclosure includes both solvated and unsolvated forms of compounds ofthe invention. One type of solvate is a hydrate. A “hydrate” relates toa particular subgroup of solvates where the solvent molecule is water.Solvates typically can function as pharmacological equivalents.Preparation of solvates is known in the art. See, e.g., M. Caira et al.,J. Pharmaceut. Sci., 93(3):601-611 (2004); E. C. van Tonder et al., AAPSPharm. Sci. Tech. 5(1): Article 12 (2004); and A. L. Bingham et al.,Chem. Commun. 603-604 (2001). A typical, non-limiting, process ofpreparing a solvate would involve dissolving a compound of the inventionin a desired solvent (organic, water, or a mixture thereof) attemperatures about 20° C. to about 25° C., then cooling the solution ata rate sufficient to form crystals, and isolating the crystals by knownmethods, e.g., filtration. Analytical techniques such as infraredspectroscopy can be used to confirm the presence of the solvent in acrystal of the solvate.

The term “prodrug” refers to a compound, which is an inactive precursorof a compound, converted into its active form in the body by normalmetabolic processes. Prodrug design is discussed generally in Hardma, etal. (Eds.), Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion isprovided in Higuchi, et al., Prodrugs as Novel Delivery Systems, Vol.14, ASCD Symposium Series, and in Roche (ed.), Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press(1987). To illustrate, prodrugs can be converted into apharmacologically active form through hydrolysis of, for example, anester or amide linkage, thereby introducing or exposing a functionalgroup on the resultant product. The prodrugs can be designed to reactwith an endogenous compound to form a water-soluble conjugate thatfurther enhances the pharmacological properties of the compound, forexample, increased circulatory half-life. Alternatively, prodrugs can bedesigned to undergo covalent modification on a functional group with,for example, glucuronic acid, sulfate, glutathione, amino acids, oracetate. The resulting conjugate can be inactivated and excreted in theurine, or rendered more potent than the parent compound. High molecularweight conjugates also can be excreted into the bile, subjected toenzymatic cleavage, and released back into the circulation, therebyeffectively increasing the biological half-life of the originallyadministered compound. Prodrugs of the compounds of the invention areintended to be covered within the scope of this invention.

The instant invention also includes the compounds which differ only inthe presence of one or more isotopically enriched atoms, for example,replacement of hydrogen with deuterium or tritium, or the replacement ofa carbon by ¹³C- or ¹⁴C-enriched carbon. The compounds of the presentinvention may also contain unnatural proportions of atomic isotopes atone or more of atoms that constitute such compounds. For example, thecompounds may be radiolabeled with radioactive isotopes, such as forexample tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopicvariations of the compounds of the present invention, whetherradioactive or not, are encompassed within the scope of the presentinvention.

The present disclosure further encompasses salts of the compounds of theinvention, including non-toxic pharmaceutically acceptable salts.Examples of pharmaceutically acceptable addition salts include inorganicand organic acid addition salts and basic salts. The pharmaceuticallyacceptable salts include, but are not limited to, metal salts such assodium salt, potassium salt, cesium salt and the like; alkaline earthmetals such as calcium salt, magnesium salt and the like; organic aminesalts such as triethylamine salt, pyridine salt, picoline salt,ethanolamine salt, triethanolamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt and the like; inorganic acid saltssuch as hydrochloride, hydrobromide, phosphate, sulphate and the like;organic acid salts such as citrate, lactate, tartrate, maleate,fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate,oxalate, formate, succinates, palmoates, benzoates, salicylates,ascorbates, glycerophosphates, ketoglutarates and the like; sulfonatessuch as methanesulfonate, benzenesulfonate, p-toluenesulfonate and thelike; salts of natural amino acids such as glycine, alanine, valine,leucine, isoleucine, norleucine, tyrosine, cystine, cysteine,methionine, proline, hydroxy proline, histidine, omithine, lysine,arginine, and serine; and salts of non-natural amino acids such asD-isomers or substituted amino acids; salts of guanidine; and salts ofsubstituted guanidine wherein the substituents are selected from nitro,amino, alkyl, alkenyl, alkynyl, ammonium or substituted ammonium saltsand aluminum salts.

The term “selective inhibitor” as applied to a biologically active agentrefers to the agent's ability to selectively reduce the target signalingactivity as compared to off-target signaling activity, via direct orindirect interaction with the target.

The term “PI3Kδ selective inhibitor” refers to Compound A as definedherein, which selectively inhibits the activity of the PI3K S isoformmore effectively than other isoforms of the PI3K family (α, β, and γ).For instance, a compound of formula A can be a compound that exhibits a50% inhibitory concentration (IC₅₀) with respect to the S typePI3-kinase that is at least 20-fold lower than the inhibitor's IC₅₀ withrespect to the rest of the other PI3K isoforms (i.e., α, β, and γ).

Inhibition of PI3K S may be of therapeutic benefit in treatment ofvarious conditions, e.g., conditions characterized by an inflammatoryresponse including but not limited to autoimmune diseases, allergicdiseases, and arthritic diseases. Importantly, inhibition of PI3K Sfunction does not appear to affect biological functions such asviability and fertility.

“Inflammatory response” as used herein is characterized by redness,heat, swelling and pain (i.e., inflammation) and typically involvestissue injury or destruction. An inflammatory response is usually alocalized, protective response elicited by injury or destruction oftissues, which serves to destroy, dilute or wall off (sequester) boththe injurious agent and the injured tissue. Inflammatory responses arenotably associated with the influx of leukocytes and/or leukocyte (e.g.,neutrophil) chemotaxis. Inflammatory responses can result from infectionwith pathogenic organisms and viruses, noninfectious means such astrauma or reperfusion following myocardial infarction or stroke, immuneresponses to foreign antigens, and autoimmune diseases. Inflammatoryresponses amenable to treatment with the methods and compounds accordingto the invention encompass conditions associated with reactions of thespecific defense system as well as conditions associated with reactionsof the non-specific defense system.

The therapeutic methods of the invention include methods for thetreatment of conditions associated with inflammatory cell activation.“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatibility antigens or cell adhesion molecules) ininflammatory cells (including, but not limited to, monocytes,macrophages, T lymphocytes, B lymphocytes, granulocytes(polymorphonuclear leukocytes including neutrophils, basophils, andeosinophils) mast cells, dendritic cells, Langerhans cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory condition.

The term “autoimmune disease” as used herein refers to any group ofdisorders in which tissue injury is associated with humoral orcell-mediated responses to the body's own constituents.

The term “transplant rejection” as used herein refers to an immuneresponse directed against grafted tissue (including organs or cells,e.g., bone marrow, characterized by a loss of function of the graftedand surrounding tissues, pain, swelling, leukocytosis, andthrombocytopenia).

The term “allergic disease” as used herein refers to any symptoms,tissue damage, or loss of tissue function resulting from allergy.

The term “arthritic disease” as used herein refers to any disease thatis characterized by inflammatory lesions of the joints attributable to avariety of etiologies.

The term “dermatitis” as used herein refers to any of a large family ofdiseases of the skin that are characterized by inflammation of the skinattributable to a variety of etiologies.

The term “synergistic effect,” as used herein, refers to agreater-than-additive therapeutic effect produced by a combination ofcompounds wherein the therapeutic effect obtained with the combinationexceeds the additive effects that would otherwise result from individualadministration the compounds alone. Embodiments of the invention includemethods of producing a synergistic effect in the treatment ofhematological cancer, wherein said effect is at least 5%, at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 200%, atleast 500%, or at least 1000% greater than the corresponding additiveeffect.

“Therapeutic synergy,” as used herein, means that a combination of ananti-CD20 antibody with Compound A produce a therapeutic effect intreatment which is greater than the additive effects of the anti-CD20antibody the PI3Kδ selective inhibitor when each is used alone.

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

II. PI3K Selective Inhibitor

As provided herein the PI3Kδ selective inhibitor used in combinationwith anti-CD20 antibodies and antigen-binding fragments thereof is acompound of formula A:

and a stereoisomer thereof, a tautomer thereof, pharmaceuticallyacceptable salts, solvates, and prodrugs thereof.

In one embodiment, Compound A used in combination with anti-CD20antibodies and antigen-binding fragments thereof, is(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,pharmaceutically acceptable salts, solvates, and prodrugs thereof. Thisstereoisomer is also referred to herein as “S-isomer of a compound offormula A,” “S-isomer,” “TGR-1202” and “RP 5307.”

In another embodiment, Compound A used in combination with anti-CD20antibodies and antigen-binding fragments thereof, is(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,pharmaceutically acceptable salts, solvates, and prodrugs thereof.

The chemical structures of these compounds are shown below:

III. Anti-CD20 Antibodies

CD20 is a tetraspanning transmembrane phospho-protein that is expressedpredominantly in pre-B cells and in mature peripheral B cells in humansand mice. In humans, CD20 is also strongly and homogeneously expressedon most mature B-cell malignancies.

As provided herein, anti-CD20 antibodies and antigen-binding fragmentsthereof can be used in combination with PI3Kδ selective inhibitor.

A number of anti-CD20 antibodies are known, including for example,ublituximab rituximab, ofatumumab (HuMax; Intracel), ocrelizumab,veltuzumab, GA101 (obinutuzumab), AME-133v (Applied MolecularEvolution), ocaratuzumab (Mentrik Biotech), PRO131921, tositumomab,ibritumomab-tiuxetan, hA20 (Immunomedics, Inc.), BLX-301 (BiolexTherapeutics), Reditux (Dr. Reddy's Laboratories), and PR070769(described in WO2004/056312).

Ublituximab (Utuxin™, LFB-R603, TG20, EMAB603) is a monoclonal antibodythat targets a specific and unique epitope on CD20 and that has beenbioengineered for enhanced clinical activity and potency.

Rituximab is a genetically engineered chimeric murine/human monoclonalantibody directed against the CD20 antigen. Rituximab is the antibodycalled “C2B8” in U.S. Pat. No. 5,736,137. The amino acid sequence ofrituximab antibody and exemplary methods for its production viarecombinant expression in Chinese Hamster Ovary (CHO) cells aredisclosed in U.S. Pat. No. 5,736,137, which is herein incorporated byreference in its entirety.

Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody. Studiesindicate that ofatumumab dissociates from CD20 at a slower rate comparedto the rituximab and binds a membrane-proximal epitope. Zhang et al.,Mabs 1. 326-331 (2009). Epitope mapping has indicated that ofatumumabbinds an epitope located closer to the N-terminus of CD20 compared tothe location targeted by rituximab and includes an extracellular loop ofthe antigen. Id.

Thus, in some embodiments, the anti CD-20 antibody or fragment thereofis selected from the group consisting of antibodies that bind to thesame epitope as ublituximab rituximab, ofatumumab, ocrelizumab,veltuzumab, GA101, AME-133v, PRO131921, tositumomab, hA20, or PR070769.In some embodiments, the anti-CD20 antibody or fragment thereof isublituximab rituximab, ofatumumab, ocrelizumab, veltuzumab, GA101,AME-133v, PRO131921, tositumomab, hA20, PR070769, or a fragment thereof.

In some embodiments, the anti-CD20 antibody or fragment thereof binds tothe same epitope as ublituximab. In some embodiments, the anti-CD20antibody or fragment thereof binds to a sequence comprising amino acidsN153-S179 of CD20. In some embodiments, the anti-CD20 antibody orfragment thereof binds to a discontinuous epitope in amino acidsN153-S179 of CD20.

In some embodiments, the anti-CD20 antibody or fragment thereof binds toCD20 with an affinity characterized by a dissociation constant KD ofless than about 10⁻⁷ M, less than about 10⁻⁸ M or less than about 10⁻⁹M. In some embodiments, the anti-CD20 antibody or fragment thereof bindsto CD20 with an affinity characterized by a dissociation constant KD of10⁻¹⁰ to 10⁻⁹ M. In some embodiments, the anti-CD20 antibody or fragmentthereof binds to CD20 with an affinity characterized by a dissociationconstant KD of 0.7×10⁻⁹ M. As used in the context of antibody bindingdissociation constants, the term “about” allows for the degree ofvariation inherent in the methods utilized for measuring antibodyaffinity. For example, depending on the level of precision of theinstrumentation used, standard error based on the number of samplesmeasured, and rounding error, the term “about 10−2 M” might include, forexample, from 0.05 M to 0.005 M.

In some embodiments, the anti-CD20 antibody exhibits a high affinity toFc-gammaRIII (CD16). In some embodiments, as a result of their highaffinity for the Fc region of the antibody to CD16, such antibodies arenot displaced by IgG polyclonal antibodies, especially by IgG present inblood serum. In some embodiments the antibody binds to CD16 (e.g.,expressed on a macrophage) with an affinity of at least 2×10⁶ M⁻¹, atleast 2×10⁷ M⁻¹, 2×10⁸ M⁻¹ or 2×10⁷ M⁻¹, e.g., as determined byScatchard analysis or BIAcore technology (Label-free surface plasmonresonance based technology).

In some embodiments, the anti-CD20 antibody exhibits a glycosylationpattern characterized by low fucose content in its Fc region. Forexample, in some embodiments, a composition comprises anti-CD20antibodies in which the antibodies comprise N-glycoside-linked sugarchains bound on the Fc-gamma glycosylation site (Asn 297, EU numbering),wherein among the N-glycoside-linked sugar chains of all the antibodiesof the composition, the fucose content is less than 65%, less than 60%,less than 55%, less than 50%, less than 45%, or less than 40%. In someembodiments, among the N-glycoside-linked sugar chains of all theantibodies of the composition, the fucose content is 15 to 45% or 20 to40%.

In some embodiments, the anti-CD20 antibody exhibits potent in vitroantibody-dependent cellular cytotoxicity (ADCC). In some embodiments,the anti-CD20 antibody produces an ADCC plateau of at least about 10%,at least about 15%, at least about 20%, at least about 25%, or at leastabout 30% at a concentration of 50 ng/ml using natural killer (NK) cellsfrom healthy donors. Techniques for measuring ADCC are known in the artand provided, for example, in de Romeauf et al., British Journal ofHaematology 140: 635-643 (2008). In some embodiments, the anti-CD20antibody produces an ADCC plateau at about 35% at a concentration of 50ng/ml using NK cells from healthy donors.

In some embodiments, the anti-CD20 antibody can decrease NF-kappa-Bactivity. In some embodiments, the anti-CD20 antibody can decrease SNAILexpression. In some embodiments, the anti-CD20 antibody can increaseRKIP activity. In some embodiments, the anti-CD20 antibody can increasePTEN activity. In some embodiments, the anti-CD20 antibody can increasesensitization of a cell to TRAIL-apoptosis.

In some embodiments, the anti-CD20 antibody is Fc-gamma-RIIIA (CD16)optimized. Antibodies capable of activating type III Fc receptors andhaving a particular glycan structure have been described, for example,in U.S. Pat. No. 7,931,895, which is herein incorporated by reference inits entirety. Thus, in some embodiments, the anti-CD20 antibody ismodified on Asn 297 (EU numbering) with N-glycosylations of thebi-antennary and/or oligomannoside type as described in U.S. Pat. No.7,931,895. Methods of producing antibodies with strong affinity forreceptor CD16 of the effector cells of the immune system are provided,for example, in U.S. Published Application No. 2005/0271652, which isherein incorporated by reference in its entirety.

In some embodiments, the anti-CD20 antibody has high ADCC activity.Methods of producing antibodies with high ADCC activity are provided,for example, in U.S. Pat. No. 7,713,524, which is herein incorporated byreference in its entirety.

Ublituximab comprises the antibody sequences provided below:

Variable heavy chain CDR1:  (SEQ ID NO: 1)Gly Tyr Thr Phe Thr Ser Tyr Asn  Variable heavy chain CDR2: (SEQ ID NO: 2) Ile Tyr Pro Gly Asn Gly Asp Thr Variable heavy chain CDR3:   (SEQ ID NO: 3)Ala Arg Tyr Asp Tyr Asn Tyr Ala Met Asp Tyr Variable heavy chain: (SEQ ID NO: 4)Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Met Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Arg Gln GlyLeu Glu Trp Ile Gly Gly Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys GlyLys Ala Thr Leu Thr Val Gly Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Val Tyr Phe Cys Ala Arg Tyr Asp Tyr Asn Tyr Ala Met Asp Tyr Trp GlyGln Gly Thr Ser Val Thr Val Ser Ser  Constant heavy chain: (SEQ ID NO: 5)Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SerGly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser LeuSer Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro LysPro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala ProIle Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr ProSer Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro Gly Lys  Variable light chain CDR1: (SEQ ID NO: 6) Ser Ser Val Ser Tyr Variable light chain CDR2:  (SEQ ID NO: 7) Ala Thr Ser Variable light chain CDR3:  (SEQ ID NO: 8)Gln Gln Trp Thr Phe Asn Pro Pro Thr  Variable light chain: (SEQ ID NO: 9)Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met ThrCys Arg Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro LysPro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly SerGly Thr Ser Tyr Ser Phe Thr Ile Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys GlnGln Trp Thr Phe Asn Pro Pro Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Constant light chain:  (SEQ ID NO: 10)Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr AlaSer Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val AspAsn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys GluVal Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 

Thus, in some embodiments, an isolated antibody or antigen-bindingfragment, variant, or derivative thereof comprises, consists essentiallyof, or consists of an immunoglobulin heavy chain variable domain (VHdomain), wherein at least one (i.e., one, two, or three) of the CDRs ofthe VH domain has an amino acid sequence that is at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or identical to the CDR1, CDR2 or CDR3 regionof sequences SEQ ID NO:1, 2, or 3, wherein an antibody orantigen-binding fragment thereof comprising the VH domain canspecifically or preferentially bind to CD20.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of an immunoglobulin heavy chain variable domain (VH domain),wherein at least one (i.e., one, two, or three) of the CDRs of the VHdomain has an amino acid sequence identical, except for 1, 2, 3, 4, or 5conservative amino acid substitutions, to the CDR1, CDR2 or CDR3 regionof sequences SEQ ID NO:1, 2, or 3, wherein an antibody orantigen-binding fragment, variant, or derivative thereof comprising theVH domain can specifically or preferentially bind to CD20.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of a VH domain that has an amino acid sequence that is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a VH amino acid sequence of SEQ ID NO:4, wherein anantibody or antigen-binding fragment, variant, or derivative thereofcomprising the VH domain can specifically or preferentially bind toCD20.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of a heavy chain that has an amino acid sequence that is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to a heavy chain amino acid sequence comprising SEQ IDNOs: 4 and 5, wherein an antibody or antigen-binding fragment, variant,or derivative thereof comprising the heavy chain can specifically orpreferentially bind to CD20.

In some embodiments, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of an immunoglobulin light chain variable domain (VL domain),wherein at least one (i.e., one, two, or three) of the CDRs of the VLdomain has an amino acid sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or identical to the CDR1, CDR2 or CDR3 region ofsequences SEQ ID NO:6, 7, or 8, wherein an antibody or antigen-bindingfragment thereof comprising the VL domain can specifically orpreferentially bind to CD20.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of an immunoglobulin light chain variable domain (VL domain),wherein at least one (i.e., one, two, or three) of the CDRs of the VLdomain has an amino acid sequence identical, except for 1, 2, 3, 4, or 5conservative amino acid substitutions, to the CDR1, CDR2 or CDR3 regionof SEQ ID NO:6, 7, or 8, wherein an antibody or antigen-bindingfragment, variant, or derivative thereof comprising the VL domain canspecifically or preferentially bind to CD20.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of a VL domain that has an amino acid sequence that is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a VL amino acid sequence of SEQ ID NO:9, wherein anantibody or antigen-binding fragment, variant, or derivative thereofcomprising the VL domain can specifically or preferentially bind toCD20.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of a light chain that has an amino acid sequence that is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to a heavy chain amino acid sequence comprising SEQ IDNOs:9 and 10, wherein an antibody or antigen-binding fragment, variant,or derivative thereof comprising the light chain can specifically orpreferentially bind to CD20.

In some embodiments, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of an immunoglobulin heavy chain variable domain (VH domain)and an immunoglobulin light chain variable domain (VL domain), whereinat least one (i.e., one, two, or three) of the CDRs of the VH domain hasan amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or identical to the CDR1, CDR2 or CDR3 region of sequences SEQID NO:1, 2, or 3, wherein at least one (i.e., one, two, or three) of theCDRs of the VL domain has an amino acid sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or identical to the CDR1, CDR2 orCDR3 region of sequences SEQ ID NO:6, 7, or 8, and wherein an antibodyor antigen-binding fragment thereof comprising the VH domain and VL canspecifically or preferentially bind to CD20.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of an immunoglobulin heavy chain variable domain (VH domain),and an immunoglobulin light chain variable domain (VL domain), whereinat least one (i.e., one, two, or three) of the CDRs of the VH domain hasan amino acid sequence identical, except for 1, 2, 3, 4, or 5conservative amino acid substitutions, to the CDR1, CDR2 or CDR3 regionof sequences SEQ ID NO:1, 2, or 3, wherein at least one (i.e., one, two,or three) of the CDRs of the VL domain has an amino acid sequenceidentical, except for 1, 2, 3, 4, or 5 conservative amino acidsubstitutions, to the CDR1, CDR2 or CDR3 region of SEQ ID NO:6, 7, or 8,and wherein an antibody or antigen-binding fragment, variant, orderivative thereof comprising the VH and VL can specifically orpreferentially bind to CD20.

In some embodiments, the anti-CD20 antibody or antigen-binding fragment,variant, or derivative thereof comprises the VH CDR1, CDR2 and CDR3region of sequences SEQ ID NO:1, 2, and 3, and the VL CDR1, CDR2 andCDR3 region of sequences SEQ ID NO:6, 7, and 8.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of a VH domain and a VL domain, wherein the VH has an aminoacid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to a VH amino acid sequence of SEQID NO:4, wherein the VL domain that has an amino acid sequence that isat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to a VL amino acid sequence of SEQ ID NO:9, and whereinan antibody or antigen-binding fragment, variant, or derivative thereofcomprising the VH and VL domain can specifically or preferentially bindto CD20.

In some embodiments, the anti-CD20 antibody or antigen-binding fragmentthereof comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:9.

In some embodiments, the anti-CD20 antibody or antigen-binding fragmentthereof binds to the same epitope as an antibody comprising the VH ofSEQ ID NO:4 and the VL of SEQ ID NO:9.

In another embodiment, an isolated antibody or antigen-binding fragment,variant, or derivative thereof comprises, consists essentially of, orconsists of a heavy chain and a light chain, wherein the heavy chain hasan amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a heavy chain aminoacid sequence comprising SEQ ID NOs: 4 and 5, wherein the light chainthat has an amino acid sequence that is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a heavychain amino acid sequence comprising SEQ ID NOs: 9 and 10, and whereinan antibody or antigen-binding fragment, variant, or derivative thereofcomprising the heavy chain can specifically or preferentially bind toCD20.

In some embodiments, the anti-CD20 antibody or antigen-binding fragmentthereof comprises a heavy chain comprising SEQ ID NOs: 4 and 5 and alight chain comprising SEQ ID NOs: 9 and 10.

In some embodiments, the anti-CD20 antibody or antigen-binding fragmentthereof binds to the same epitope as an antibody comprising SEQ ID NO:4and SEQ ID NO:5.

In some embodiments, the anti-CD20 antibody is ublituximab.

In some embodiments, the antibody is EMAB603 (see WO2006/064121, whichis herein incorporated by reference in its entirety), produced by theclone R603-12D11, deposited to the Collection Nationale des Cultures deMicroorganismes under the accession number CNCM I-3529.

In some embodiments, the anti-CD20 antibody is produced in the rathybridoma YB2/0 cell line (cell YB2/3HL.P2.G11.16Ag.20, registered atthe American Type Culture Collection under ATCC number CRL-1662).

The precise chemical structure of an antibody capable of specificallybinding CD20 and retaining the desired activity depends on a number offactors. As ionizable amino and carboxyl groups are present in themolecule, a particular polypeptide can be obtained as an acidic or basicsalt, or in neutral form. All such preparations that retain theirbiological activity when placed in suitable environmental conditions areincluded in the definition of anti-CD20 antibodies as used herein.Further, the primary amino acid sequence of the antibody can beaugmented by derivatization using sugar moieties (glycosylation) or byother supplementary molecules such as lipids, phosphate, acetyl groupsand the like. It can also be augmented by conjugation with saccharides.Certain aspects of such augmentation are accomplished throughpost-translational processing systems of the producing host; other suchmodifications can be introduced in vitro. In any event, suchmodifications are included in the definition of an anti-CD20 antibodyused herein so long as the desired properties of the anti-CD20 antibodyare not destroyed. It is expected that such modifications canquantitatively or qualitatively affect the activity, either by enhancingor diminishing the activity of the polypeptide, in the various assays.Further, individual amino acid residues in the chain can be modified byoxidation, reduction, or other derivatization, and the polypeptide canbe cleaved to obtain fragments that retain activity. Such alterationsthat do not destroy the desired properties (e.g., binding specificityfor CD20) do not remove the polypeptide sequence from the definition ofanti-CD20 antibodies of interest as used herein.

The art provides substantial guidance regarding the preparation and useof polypeptide variants. In preparing variants of an anti-CD20 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, one of skill in the art can readily determine whichmodifications to the native protein's nucleotide or amino acid sequencewill result in a variant that is suitable for use as a therapeuticallyactive component of a pharmaceutical composition.

It is possible to introduce mutations only in framework regions or onlyin CDR regions of an antibody molecule. Introduced mutations can besilent or neutral missense mutations, i.e., have no, or little, effecton an antibody's ability to bind antigen. These types of mutations canbe useful to optimize codon usage, or improve a hybridoma's antibodyproduction. Alternatively, non-neutral missense mutations can alter anantibody's ability to bind antigen. The location of most silent andneutral missense mutations is likely to be in the framework regions,while the location of most non-neutral missense mutations is likely tobe in CDR, though this is not an absolute requirement. One of skill inthe art would be able to design and test mutant molecules with desiredproperties such as no alteration in antigen-binding activity oralteration in binding activity (e.g., improvements in antigen-bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein can routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of a CD20 polypeptide) canbe determined using techniques described herein or by routinelymodifying techniques known in the art.

In certain embodiments, the anti-CD20 antibodies comprise at least oneoptimized complementarity-determining region (CDR). By “optimized CDR”is intended that the CDR has been modified and optimized sequencesselected based on the sustained or improved binding affinity and/oranti-CD20 activity that is imparted to an anti-CD20 antibody comprisingthe optimized CDR. “Anti-CD20 activity” can include, e.g., activitywhich modulates one or more of the following activities associated withCD20, e.g., the ability to induce apoptosis of B-cells, the ability toinduce ADCC against B-cells (e.g., CLL cells), the ability to inhibitNF-kappaB activity, the ability to inhibit Snail expression, the abilityto de-repress RKIP, the ability to de-repress PTEN, the ability tosensitize a tumor cell to TRAIL-apoptosis or any other activityassociated with CD20. Such activities are described, for example, inBaritaki et al., International Journal of Oncology 38: 1683-1694 (2011),which is herein incorporated by reference in its entirety. Themodifications can involve replacement of amino acid residues within theCDR such that an anti-CD20 antibody retains specificity for the CD20antigen and has improved binding affinity and/or improved anti-CD20activity.

In certain anti-CD20 antibodies, or antigen-binding fragments thereof,at least a fraction of one or more of the constant region domains hasbeen deleted or otherwise altered so as to provide desired biochemicalcharacteristics such as reduced effector functions, the ability tonon-covalently dimerize, increased ability to localize at the site of atumor, reduced serum half-life, or increased serum half-life whencompared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the CH₂ domain will be deleted.

In certain anti-CD20 antibodies or antigen-binding fragments thereof,the Fc portion can be mutated to decrease effector function usingtechniques known in the art. For example, modifications of the constantregion can be used to modify disulfide linkages or oligosaccharidemoieties that allow for enhanced localization due to increased antigenspecificity or antibody flexibility. The resulting physiologicalprofile, bioavailability and other biochemical effects of themodifications can easily be measured and quantified using well knowimmunological techniques without undue experimentation.

In certain embodiments, an anti-CD20 antibody or antigen-bindingfragment thereof will not elicit a deleterious immune response in theanimal to be treated, e.g., in a human. In one embodiment, anti-CD20antibodies or antigen-binding fragments thereof can be modified toreduce their immunogenicity using art-recognized techniques. Forexample, antibodies can be humanized, primatized, deimmunized, orchimeric antibodies can be made. These types of antibodies are derivedfrom a non-human antibody, typically a murine or primate antibody, thatretains or substantially retains the antigen-binding properties of theparent antibody, but which is less immunogenic in humans. This can beachieved by various methods, including (a) grafting the entire non-humanvariable domains onto human constant regions to generate chimericantibodies; (b) grafting at least a part of one or more of the non-humancomplementarity determining regions (CDRs) into a human framework andconstant regions with or without retention of critical frameworkresidues; or (c) transplanting the entire non-human variable domains,but “cloaking” them with a human-like section by replacement of surfaceresidues. Such methods are disclosed in Morrison et al., Proc. Natl.Acad. Sci. 81:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92(1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec.Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), andU.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all ofwhich are hereby incorporated by reference in their entirety.

Modified forms of antibodies or antigen-binding fragments thereof can bemade from whole precursor or parent antibodies using techniques known inthe art.

Anti-CD20 antibodies or antigen-binding fragments thereof can be made ormanufactured using techniques that are known in the art. In certainembodiments, antibody molecules or fragments thereof are “recombinantlyproduced,” i.e., are produced using recombinant DNA technology.Anti-CD20 antibodies or fragments thereof can be generated by anysuitable method known in the art including generation of polyclonalantibodies or preparation of monoclonal antibodies, e.g., throughhybridoma or phage display.

A variety of host-expression vector systems can be utilized to expressantibody molecules. The host cell can be co-transfected with twoexpression vectors, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector can be used which encodes both heavy andlight chain polypeptides. In such situations, the light chain isadvantageously placed before the heavy chain to avoid an excess of toxicfree heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.Acad. Sci. USA 77:2197 (1980)). The host cell can also be transfectedwith a single vector encoding a heavy chain derived polypeptide and alight chain derived polypeptide. The coding sequences for the heavy andlight chains can comprise cDNA or genomic DNA.

The expression vector or vectors can be transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody. Thus, host cellscontaining a polynucleotide encoding an antibody, or a heavy or lightchain thereof, operably linked to a heterologous promoter are provided.In certain embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains can be co-expressed inthe host cell for expression of the entire immunoglobulin molecule.

Host-expression systems represent vehicles by which the coding sequencesof interest can be produced and subsequently purified, but alsorepresent cells which can, when transformed or transfected with theappropriate nucleotide coding sequences, express a CD20 antibody insitu. These include but are not limited to microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining antibody coding sequences; yeast (e.g., Saccharomyces,Pichia) transformed with recombinant yeast expression vectors containingantibody coding sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Bacterial cells such as Escherichia coli, or eukaryoticcells, e.g., for the expression of whole recombinant antibody molecules,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese hamster ovary cells (CHO), inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking et al., Gene 45:101 (1986); Cockett etal., Bio/Technology 8:2 (1990)). In some embodiments, the anti-CD20antibody is produced in a host cell that is not a CHO cell.

Once an antibody has been recombinantly expressed, it can be purified byany method known in the art for purification of an immunoglobulinmolecule, for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.

In some embodiments, the anti-CD20 antibody is produced by a rathybridoma cell line. In some embodiments, the anti-CD20 antibody isproduced in YB2/0 (ATCC CRL-1662)

IV. Pharmaceutical Compositions

A compound of formula A and anti-CD20 antibodies can be administered inany order or at any interval as determined by one of skill in the art.For example, a compound of formula A and anti-CD20 antibody can beadministered sequentially (in any order), simultaneously, or via anycombination of sequential and simultaneous administrations. A compoundof formula A and anti-CD20 antibody can be administered in the samepharmaceutical compositions or in separate pharmaceutical compositions.

Administration of combination, whether simultaneous, sequential (in anyorder) or both, can be performed according to any number of desiredintervals of minutes (e.g., 0-60 minutes), hours (e.g., 0-24 hours),days (e.g., 0-7 days), and/or weeks (e.g., 0-52 weeks) as can be decidedand determined by one of skill in the art. The dosing can also vary overtime, for example, starting with a once weekly dose for a period of time(e.g., for 1, 2, 3, 4, 5, or 6 weeks) followed by dosing once every twoweeks, once every three weeks, once every four weeks, once every fiveweeks, or once every six weeks.

The Compound of formula A and anti-CD20 antibodies can be formulatedinto pharmaceutical compositions for administration to mammals,including humans. The pharmaceutical compositions comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions can be administered by any suitable method, e.g.,parenterally, intraventricularly, orally, by inhalation spray,topically, rectally, nasally, buccally, vaginally or via an implantedreservoir. In some embodiments, the Compound of formula A isadministered orally. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

Parenteral formulations can be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionscan be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis. In some embodiments, the anti-CD20antibody is administered intravenously (IV).

Certain pharmaceutical compositions can be orally administered in anacceptable dosage form including, e.g., capsules, tablets, aqueoussuspensions or solutions. Certain pharmaceutical compositions also canbe administered by nasal aerosol or inhalation. Such compositions can beprepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,and/or other conventional solubilizing or dispersing agents.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular therapeuticagents used, the patient's age, body weight, general health, sex, anddiet, and the time of administration, rate of excretion, drugcombination, and the severity of the particular disease being treated.Judgment of such factors by medical caregivers is within the ordinaryskill in the art. The amount will also depend on the individual patientto be treated, the route of administration, the type of formulation, thecharacteristics of the compound used, the severity of the disease, andthe desired effect. The amount used can be determined by pharmacologicaland pharmacokinetic principles well known in the art.

In some embodiments, the anti-CD20 antibody is administered at a dose ofless than 187.5 mg/m², 75 mg/m², 37.5 mg/m², 15 mg/m², 7.5 mg/m², 3.75mg/m². In some embodiments, the dose administered can be 187.5 mg/m² to75 mg/m², 75 mg/m² to 37.5 mg/m², 75 mg/m² to 15 mg/m², 75 mg/m² to 7.5mg/m², or 75 mg/m² to 3.75 mg/m².

In some embodiments, a compound of formula A is administered at a doserange of 10 to 2500 mg/day, 10 to 1500 mg/day, 50 to 1000 mg/day, 100 to750 mg mg/day, 150 to 500 mg/day per day. In some embodiments, the doseadministered can be 200 to 400 mg per day. In some embodiments, the doseadministered can be 500, 1000, 1500, 2000 or 2500 mg/day.

Supplementary active compounds also can be incorporated into thecompositions. For example, an anti-CD20 antibody and a Compound offormula A can be coformulated with and/or coadministered with one ormore additional therapeutic agents, such as anti-cancer agents.

V. Kits

The present invention provides kits that comprise a compound of formulaA, anti-CD20 antibodies, other agents and that can be used to performthe methods described herein, and combinations thereof. In certainembodiments, a kit comprises at least one purified antibody against CD20in one or more containers and instructions for using the antibody incombination with a compound of formula A. In certain embodiments, a kitcomprises a compound of formula A and instructions for using theinhibitor in combination with an anti-CD20 antibody. In certainembodiments, a kit comprises at least one anti-CD20 antibody and acompound of formula A.

Pharmaceutical kits comprising one or more containers filled with one ormore of the ingredients of the pharmaceutical compounds and compositionsof the present invention, including, a compound of formula A and/or oneor more anti-CD20 antibodies are also provided herein. Such kits canalso include, for example, other compounds and/or compositions, adevice(s) for administering the compounds and/or compositions, andwritten instructions in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts

One skilled in the art will readily recognize that the disclosedantibodies and a compound of formula A described herein can be readilyincorporated into one of the established kit formats which are wellknown in the art.

Further provided are kits comprising a (a) a compound of formula A, ananti-CD20 antibody, or a combination thereof and (b) an additionalanti-cancer agent. In certain embodiments, the additional anti-canceragent is a chemotherapeutic agent.

VI. Methods of Using Combinations of a Compound of Formula A andAnti-CD20 Antibodies

Combinations of a compound of formula A and anti-CD20 antibodies can beused in methods of treating diseases or disorders in a subject.

Thus, uses of a compound of formula A in the manufacture of a medicamentfor the treatment of a proliferative disorder wherein the a compound offormula A is to be administered in combination (e.g., sequentially orsimultaneously) with an anti-CD20 antibody are provided. In addition,uses of an anti-CD20 antibody in the manufacture of a medicament for thetreatment of a proliferative disorder wherein the anti-CD20 antibody isto be administered in combination (e.g., sequentially or simultaneously)with a compound of formula A are also provided.

The invention further provides a method of inhibiting PI3Kδ isoformand/or CD20 in a patient by administering to a patient an effectiveamount of a combination of the present invention.

The invention further provides a method of treating, preventing, and/orinhibiting a PI3Kδ mediated disease, disorder or condition and/or a CD20mediated disease, disorder, or condition (such as cancer or otherproliferative disease or disorder) in a patient by administering to apatient an effective amount of a combination of the present invention.

The invention further provides a method of treating a PI3Kδ isoformand/or CD20 associated disease, disorder or condition in a patient byadministering to the patient an effective amount of a combination of thepresent invention. In one embodiment, the amount of the compoundadministered in a combination is sufficient to treat a PI3Kδ isoformand/or CD20 associated disease, disorder or condition by selectiveinhibition of PI3K S and/or CD20

The invention further provides a method for treating a proliferativedisease by administering to a patient in need of such treatment aneffective amount of at least one compound of formula A and antibody ofthe present invention. In one embodiment, the amount of the compoundadministered in combination is sufficient to treat the proliferativedisease by selective inhibition of PI3K S and/or inhibition of CD20.

The invention further provides a method for treating a proliferativedisease by administering to a patient in need of such treatment aneffective amount of a combination of the present invention, in furthercombination (simultaneously or sequentially) with at least one otheranti-cancer agent. In one embodiment, the amount of the compound Aadministered is sufficient to treat (or facilitate treatment of) theproliferative disease by selective inhibition of PI3K S.

The combinations of the present invention are useful in the treatment ofa variety of cancers, including, but not limited to, the following:

-   -   carcinoma, including that of the bladder, breast, colon, kidney,        liver, lung (including small cell lung cancer), esophagus, gall        bladder, uterus, ovary, testes, larynx, oral cavity,        gastrointestinal tract (e.g., esophagus, stomach, pancreas),        brain, cervix, thyroid, prostate, blood, and skin (including        squamous cell carcinoma);    -   hematopoietic tumors of lymphoid lineage, including leukemia,        acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell        lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkins        lymphoma, hairy cell lymphoma and Burkett's lymphoma;    -   hematopoietic tumors of myeloid lineage, including acute and        chronic myelogenous leukemias, myelodysplastic syndrome and        promyelocytic leukemia;    -   tumors of mesenchymal origin, including fibrosarcoma and        rhabdomyosarcoma;    -   tumors of the central and peripheral nervous system, including        astrocytoma, neuroblastoma, glioma and schwannomas; and    -   other tumors, including melanoma, seminoma, teratocarcinoma,        osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid        follicular cancer and Kaposi's sarcoma.

The combinations of the present invention as modulators of apoptosis areuseful in the treatment, prevention, and inhibition of cancer(including, but not limited to, those types mentioned herein above).

The combinations of the present invention are useful in thechemoprevention of cancer. Chemoprevention involves inhibiting thedevelopment of invasive cancer by blocking the initiating mutagenicevent, blocking the progression of pre-malignant cells that have alreadysuffered an insult, or inhibiting tumor relapse. The compounds are alsouseful in inhibiting tumor angiogenesis and metastasis. One embodimentof the invention is a method of inhibiting tumor angiogenesis ormetastasis in a patient by administering an effective amount of one ormore compounds of the present invention.

The invention further provides a method of treating an immunesystem-related disease (e.g., an autoimmune disease), a disease ordisorder involving inflammation (e.g., asthma, chronic obstructivepulmonary disease, rheumatoid arthritis, inflammatory bowel disease,glomerulonephritis, neuroinflammatory diseases, multiple sclerosis,uveitis and disorders of the immune system), cancer or otherproliferative disease, a hepatic disease or disorder, or a renal diseaseor disorder. The method includes administering an effective amount of acombination of the present invention.

Examples of immune disorders which can be treated by the compounds ofthe present invention include, but are not limited to, psoriasis,rheumatoid arthritis, vasculitis, inflammatory bowel disease,dermatitis, osteoarthritis, asthma, inflammatory muscle disease,allergic rhinitis, vaginitis, interstitial cystitis, scleroderma,osteoporosis, eczema, allogeneic or xenogeneic transplantation (organ,bone marrow, stem cells and other cells and tissues) graft rejection,graft-versus-host disease, lupus erythematosus, inflammatory disease,type I diabetes, pulmonary fibrosis, dermatomyositis, Sjogren'ssyndrome, thyroiditis (e.g., Hashimoto's and autoimmune thyroiditis),myasthenia gravis, autoimmune hemolytic anemia, multiple sclerosis,cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis,allergic conjunctivitis and atopic dermatitis.

The invention further provides a method of treating leukemia in apatient by administering a therapeutically effective amount of acombination of the present invention. For example, the methods of thepresent invention are effective for treating chronic lymphocyticleukemia (CLL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia(AML), multiple myeloma (MM), small lymphocytic lymphoma (SLL), andindolent non-Hodgkin's lymphoma (I-NHL).

In the aforementioned methods of treatment, one or more additionalactive agents can be administered with the combinations of the presentinvention. For example, the combination of the present invention areuseful in combining (administered together or sequentially) with knownanti-cancer treatments such as radiation therapy or with one or morecytostatic, cytotoxic or anticancer agents, such as, for example, DNAinteractive agents, such as cisplatin or doxorubicin; topoisomerase IIinhibitors, such as etoposide; topoisomerase I inhibitors such as CPT-11or topotecan; tubulin interacting agents, such as paclitaxel, docetaxelor the epothilones (for example ixabepilone), either naturally occurringor synthetic; hormonal agents, such as tamoxifen; thymidilate synthaseinhibitors, such as 5-fluorouracil; and anti-metabolites, such asmethotrexate; other tyrosine kinase inhibitors such as Iressa andOSI-774; angiogenesis inhibitors; EGF inhibitors; VEGF inhibitors; CDKinhibitors; SRC inhibitors; c-Kit inhibitors; Her1/2 inhibitors andmonoclonal antibodies directed against growth factor receptors such aserbitux (EGF) and herceptin (Her2); and other protein kinase modulators.The additional active agent can also be a proteasome inhibitor,Bortezomib (Velcade®), Carfilzomib (PR-171), PR-047, disulfiram,lactacystin, PS-519, eponemycin, epoxomycin, aclacinomycin, CEP-1612,MG-132, CVT-63417, PS-341, vinyl sulfone tripeptide inhibitors,ritonavir, PI-083, (+/−)-7-methylomuralide, (−)-7-methylomuralide,lenalidomide (Revlimid®), or a combination thereof.

The combinations of the present invention are also useful in combining(administered together or sequentially) with one or more steroidalanti-inflammatory drugs, non-steroidal anti-inflammatory drugs (NSAIDs)or immune selective anti-inflammatory Derivatives (ImSAIDs).

Ina particular embodiment, the cancer is a hematological malignancyand/or solid tumor. In another particular embodiment, the hematologicalmalignancy is leukemia or lymphoma.

In some embodiments, lymphoma is a mature (peripheral) B-cell neoplasm.In specific embodiments, the mature B-cell neoplasm is selected from thegroup consisting of B-cell chronic lymphocytic leukemia/smalllymphocytic lymphoma; B-cell prolymphocytic leukemia; Lymphoplasmacyticlymphoma; Marginal zone lymphoma, such as Splenic marginal zone B-celllymphoma (+/−villous lymphocytes), Nodal marginal zone lymphoma(+/−monocytoid B-cells), and Extranodal marginal zone B-cell lymphoma ofmucosa-associated lymphoid tissue (MALT) type; Hairy cell leukemia;Plasma cell myeloma/plasmacytoma; Follicular lymphoma, follicle center;Mantle cell lymphoma; Diffuse large cell B-cell lymphoma (includingMediastinal large B-cell lymphoma, Intravascular large B-cell lymphoma,and Primary effusion lymphoma); and Burkitt's lymphoma/Burkitt's cellleukemia.

In some embodiments, lymphoma is selected from the group consisting ofmultiple myeloma (MM) and non-Hodgkin's lymphoma (NHL), mantle celllymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulinemia(WM) or B-cell lymphoma and diffuse large B-cell lymphoma (DLBCL).

In a further particular embodiment, leukemia is selected from the groupconsisting of acute lymphocytic leukemia/acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),and small lymphocytic lymphoma (SLL). In some embodiments, Non-Hodgkin'sLymphoma (NHL) is aggressive NHL or indolent NHL. Examples of aggressiveNHL includes B-cell neoplasms, diffuse large B-cell lymphoma, T/NK cellneoplasms, anaplastic large cell lymphoma, peripheral T-cell lymphomas,precursor B-lymphoblastic leukemia/lymphoma, precursor T-lymphoblasticleukemia/lymphoma, Burkitt's lymphoma, Adult T-cell lymphoma/leukemia(HTLV1+), primary CNS lymphoma, mantle cell lymphoma, polymorphicpost-transplantation lymphoproliferative disorder (PTLD), AIDS-relatedlymphoma, true histiocytic lymphoma, and blastic NK-cell lymphoma. Themost common type of aggressive NHL is diffuse large cell lymphoma.Non-limiting examples of indolent NHL include follicular lymphoma, smalllymphocytic lymphoma, marginal zone lymphoma (such as extranodalmarginal zone lymphoma (also called mucosa associated lymphoidtissue—MALT lymphoma), nodal marginal zone B-cell lymphoma (monocytoidB-cell lymphoma), splenic marginal zone lymphoma), and lymphoplasmacyticlymphoma (Waldenstrom's macroglobulinemia). In some embodiments, asubject has aggressive NHL or indolent NHL.

In some embodiments, a patient has a condition selected from the groupconsisting of mantle cell lymphoma (MCL), diffuse large B cell lymphoma(DLBCL), follicular lymphoma (FL), acute lymphocytic leukemia (ALL),acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), andsmall lymphocytic lymphoma (SLL), multiple myeloma (MM), and marginalzone lymphoma.

In some embodiments, a patient has a relapsed or refractory condition.In a particular embodiment, the subject is refractory to chemotherapytreatment, or in relapse after treatment with chemotherapy.

In some embodiments, the cancer is resistant to treatment withrituximab. In some embodiments, the cancer shows a reduced response totreatment with rituximab. In some embodiments, the subject haspreviously been treated with rituximab.

In a particular embodiment, the methods comprise reducing the level ofNF-kappa-B activity, reducing SNAIL expression, increasing RKIPactivity, increasing PTEN activity, increasing tumor sensitivity toTRAIL-apoptosis, reducing the level of PI3K δ activity or a combinationthereof in a patient.

In a particular embodiment, the combination of the compound of formula Aand the anti-CD20 antibody depletes B-cells from human whole blood. Insome embodiments, the combination of the compound of formula A and theanti-CD20 antibody depletes B-cells from human whole blood to a greaterextent than either the compound of formula A or the anti-CD20 antibodyalone depletes B-cells from human whole blood. In some embodiments, thecombination of the compound of formula A and the anti-CD20 antibodydepletes B-cells from human whole blood to a greater extent than the sumof the depletion by the compound of formula A and the depletion by theanti-CD20 antibody.

In some embodiments, a compound of formula A and anti-CD20 antibody areused in a method of treating a disease or disorder associated withexcessive B-cell proliferation, wherein the method comprisesadministration of a compound of formula A and the anti-CD20 antibody toa subject in need thereof. In some embodiments, a compound of formula Aand anti-CD20 antibody are used in a method of treating a disease ordisorder associated with excessive B-cell activity, wherein the methodcomprises administration of a compound of formula A and the anti-CD20antibody to a subject in need thereof. In some embodiments, a compoundof formula A and anti-CD20 antibody are used in a method of treating adisease or disorder associated with excessive number of B-cells, whereinthe method comprises administration of a compound of formula A and theanti-CD20 antibody to a subject in need thereof.

A compound of formula A can be prepared using the general syntheticmethods as disclosed in International Patent Application Publication No.WO 2011/055215 A2 and U.S. Patent Application Publication No.2011/0118257 A1, and specific compound preparation is as disclosed inIndian provisional patent application 2693/CHE/2012 filed 4 Jul. 2012,U.S. provisional patent application U.S. Ser. No. 61/691,586 filed 21Aug. 2012, PCT/US2013/055434 filed 2 Jul. 2013 and U.S. Ser. No.13/933,856 filed 2 Jul. 2013. The entirety of each of these applicationsand publications is incorporated herein by reference.

Examples Synthesis of Compound of Formula A

Unless otherwise stated, purification implies column chromatographyusing silica gel as the stationary phase and a mixture of petroleumether (boiling at 60-80° C.) and ethyl acetate or dichloromethane andmethanol of suitable polarity as the mobile phases. The term “RT” refersto ambient temperature (25-28° C.).

Intermediate 1:2-(1-bromoethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one

Step-1

[1-(5-Fluoro-2-hydroxyphenyl)-2-(3-fluorophenyl)ethanone]:3-Fluorophenylacetic acid (7.33 g, 47.56 mmoles) was dissolved in 25 mldichloromethane. To this mixture, oxalylchloride (7.54 g, 59.46 mmoles)and DMF (3 drops) were added at 0° C. and stirred for 30 min. Thesolvent was evaporated and dissolved in 25 ml dichloromethane. To thismixture, 4-fluoroanisole (5.00 g, 39.64 mmoles) was added and cooled to0° C. At 0° C. AlCl₃ (7.95 g, 59.46 mmoles) was added and the reactionmixture was warmed to RT and stirred for 12 hours. The reaction mixturewas quenched by the addition of 2N HCl, extracted with ethyl acetate,dried over sodium sulphate and concentrated. The crude product waspurified by column chromatography with ethyl acetate:petroleum ether toafford the title compound as colorless solid (4.5 g, 45% yield). ¹H-NMR(S ppm, DMSO-D₆, 400 MHz): δ 11.34 (s, 1H), 7.75 (dd, J=9.4, 3.1 Hz,1H), 7.42 (m, 2H), 7.12 (m, 3H), 7.05 (dd, J=9.0, 4.5 Hz, 1H), 4.47 (s,2H).

Step-2

[2-Ethyl-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one]:1-(5-Fluoro-2-hydroxyphenyl)-2-(3-fluorophenyl)ethanone obtained fromStep-1 (3.00 g, 12.08 mmoles) was placed in a round bottom flask and tothis triethylamine (25 ml) and propionic anhydride (4.92 g, 37.82mmoles) were added, and the mixture was refluxed for 24 hours. Aftercooling to RT, the reaction mixture was acidified by the addition of 1NHCl solution, extracted with ethyl acetate, washed with sodiumbicarbonate solution, dried with sodium sulphate and concentrated. Thecrude product was purified by column chromatography with ethylacetate:petroleum ether to afford the title compound as off-yellow solid(1.80 g, 52% yield). ¹H-NMR (δ ppm, DMSO-D₆, 400 MHz): δ 7.80 (m, 1H),7.76 (m, 2H), 7.51 (dd, J=8.0, 6.4 Hz), 7.22 (m, 1H), 7.18 (m, 2H), 2.56(q, J=7.6 Hz, 2H), 1.20 (t, J=7.6 Hz, 3H).

Step-3:

To a solution of 2-Ethyl-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-oneobtained from Step-2 (1.80 g, 6.28 mmoles) in carbon tetrachloride (20ml), N-bromosuccinimide (1.11 g, 6.28 mmoles) was added and heated to80° C. Azobisisobutyronitrile (10 mg) was added to the reaction mixtureat 80° C. After 12 hours, the reaction mixture was cooled to RT, dilutedwith dichloromethane and washed with water. The organic layer was driedover sodium sulphate and concentrated under reduced pressure to affordthe crude title compound as yellow solid (1.25 g, 55% yield). ¹H-NMR (δppm, DMSO-D₆, 400 MHz): δ 7.91 (dd, J=9.2, 4.3 Hz, 1H), 7.81 (dt, J=8.2,2.8 Hz, 1H), 7.74 (dd, J=8.3, 3.1 Hz, 1H), 7.57 (m, 1H), 7.32 (dt,J=8.5, 2.4 Hz, 1H), 7.19 (m, 2H), 5.00 (q, J=6.8 Hz, 1H), 1.97 (d, J=6.8Hz, 3H).

Intermediate 2:6-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one

To a solution of Intermediate 1 (15.0 g, 40.84 mmol) in DMSO (150 ml),n-butanol (7.5 ml) was added and heated to 120° C. for 3 hours. Thereaction mixture was cooled to RT, quenched with water and extractedwith ethyl acetate. The organic layer was dried over sodium sulphate andconcentrated under reduced pressure. The crude product was purified bycolumn chromatography with ethyl acetate:petroleum ether to afford thetitle compound as an off-white solid (7.90 g, 64%). ¹H-NMR (δ ppm,CDCl₃, 400 MHz): 7.85 (dd, J 8.1, 3 Hz, 1H), 7.54 (dd, J 9.2, 4.2 Hz,1H), 7.47-7.37 (m, 2H), 7.15-6.98 (m, 3H), 4.74 (quintet, J 6.8 Hz, 1H),2.23 (d, J 7.4 Hz, 1H), 1.54 (d, J 6.6 Hz, 3H).

Intermediate 3: 2-acetyl-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one

DMSO (5.60 ml, 79.14 mmol) was added to dichloromethane (40 ml), andcooled to −78° C., followed by oxalyl chloride (3.40 ml, 39.57 mmol).After 10 min., intermediate 2 (6.00 g, 19.78 mmol) in dichloromethane(54 ml) was added dropwise and stirred for 20 min. Triethylamine (12 ml)was added and stirred for 1 hour. The reaction mixture was quenched withwater and extracted with dichloromethane. The organic layer was driedover sodium sulphate and concentrated under reduced pressure. The crudeproduct was purified by column chromatography with ethylacetate:petroleum ether to afford the title compound as a yellow solid(4.2 g, 71%) which was used as such in the next step.

Intermediate 4:(S)-6-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one

To intermediate 3 (2.00 g, 6.66 mmol), R-Alpine borane (0.5 M in THF, 20ml) was added and heated to 60° C. for 20 hours. The reaction mixturequenched with 2N HCl, and extracted with ethyl acetate. The organiclayer was dried over sodium sulphate and concentrated under reducedpressure. The crude product was purified by column chromatography withethyl acetate: petroleum ether to afford the title compound as anoff-white solid (1.51 g, 75%). Enantiomeric excess: 94.2%, enriched inthe fast eluting isomer (retention time: 8.78 min.) as determined byHPLC on a chiralpak AD-H column.

Intermediate 5:(R)-1-(6-fluoro-3-(3-fluorophenyl)-4-oxo-4H-chromen-2-yl)ethyl4-chlorobenzoate

To a solution of intermediate 4 (1.45 g, 4.78 mmol) in TH (15 ml),4-chlorobenzoic acid (0.748 g, 4.78 mmol) and triphenylphosphine (1.88g, 7.17 mmol) were added and heated to 45° C. followed bydiisopropylazodicarboxylate (1.4 ml, 7.17 mmol). After 1 hour, thereaction mixture was concentrated and the residue was purified by columnchromatography with ethyl acetate: petroleum ether to afford the titlecompound as an off-white solid (1.81 g, 86%) which was used withoutpurification in the next step.

Intermediate 6:(R)-6-fluoro-3-(3-fluorophenyl)-2-(1-hydroxyethyl)-4H-chromen-4-one

Method A

Intermediate 5 (1.75 g, 3.96 mmol) in methanol (17 ml) was cooled to 10°C., potassium carbonate (0.273 g, 1.98 mmol) was added and stirred for30 min. The reaction mixture was concentrated, acidified with 2N HClsolution, extracted with ethyl acetate, dried over sodium sulphate andconcentrated under reduced pressure. The crude product was purified bycolumn chromatography with ethyl acetate:petroleum ether to afford thetitle compound as a yellow solid (1.05 g, 87% yield). Enantiomericexcess: 93.6%, enriched in the late eluting isomer (retention time:11.12 min.) as determined by HPLC on a chiralpak AD-H column.

Method B

Step-1

[(R)-2-(1-(benzyloxy)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one]:To 1-(5-fluoro-2-hydroxyphenyl)-2-(3-fluorophenyl)ethanone (11.00 g,44.31 mmol) in dichloromethane, HATU (33.7 g, 88.63 mmol) andR-(+)2-benzyloxypropionic acid (9.58 g, 53.17 mmol) were added andstirred for 10 min. Triethylamine (66.7 ml, 0.47 mol) was added dropwiseand stirred at RT for 24 hours. The reaction mixture was quenched withwater, extracted with dichloromethane, dried over sodium sulphate andconcentrated under reduced pressure. The crude product was purified bycolumn chromatography with ethyl acetate:petroleum ether to afford thetitle compound as a yellow solid (10.5 g, 60% yield). ¹H-NMR (δ ppm,CDCl₃, 400 MHz): 7.85 (dd, J 8.1.3 Hz, 1H), 7.58 (dd, J 9.1, 4.1 Hz,1H), 7.47-7.39 (m, 1H), 7.39-7.34 (m, 1H), 7.28-7.20 (m, 3H), 7.20-7.14(m, 2H), 7.16-7.07 (m, 1H), 6.99-6.89 (m, 2H), 4.50-4.31 (m, 3H), 1.56(d, J 6.4 Hz, 3H).

Step-2:

(R)-2-(1-(benzyloxy)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-oneobtained in Step-1 (10.5 g, 26.69 mmol) in dichloromethane (110 ml) wascooled to 0° C., aluminium chloride (5.35 g, 40.03 mmol) was addedportionwise and stirred at RT for 6 hours. The reaction mixture wasquenched with 2N HCl solution, extracted with dichloromethane, driedover sodium sulphate and concentrated under reduced pressure. The crudeproduct was purified by column chromatography with ethylacetate:petroleum ether to afford intermediate 6 a yellow solid (6.1 g,76% yield). Enantiomeric excess: 97.7%, enriched in the late elutingisomer (retention time: 11.12 min.) as determined by HPLC on a chiralpakAD-H column.

Intermediate 7: 4-bromo-2-fluoro-1-isopropoxybenzene

To a solution of 4-bromo-3-fluorophenol (10 g, 52.35 mmol) in TH (100ml), isopropyl alcohol (4.8 ml, 62.62 mmol) and triphenylphosphine (20.6g, 78.52 mmol) were added and heated to 45° C. followed bydiisopropylazodicarboxylate (15.4 ml, 78.52 mmol). The mixture wasrefluxed for 1 hour, concentrated and the residue was purified by columnchromatography with ethyl acetate:petroleum ether to afford the titlecompound as a colorless liquid (13.1 g, 99% yield), which was usedwithout purification in the next step.

Intermediate 8:2-(3-fluoro-4-isopropoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

Potassium acetate (10.52 g, 107.2 mmol) and bis(pinacolato)diboron (15g, 58.96 mmol) were added to a solution of intermediate 7 (10.52 g,107.2 mmol) in dioxane (125 ml), and the solution was degassed for 30min. [1,1′-Bis(diphenylphosphino)ferrocene]dichloro palladium(II) CH₂Cl₂(4.4 g, 5.36 mmol) was added under nitrogen atmosphere and heated to 80°C. After 12 hours, the reaction mixture was filtered through celite andconcentrated. The crude product was purified by column chromatographywith ethyl acetate:petroleum ether to afford the title compound as ayellow oil (13.9 g, 99%) which was used without purification in the nextstep.

Intermediate 9:3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (11.0 g,42.14 mmol) in DMF (110 ml), ethanol (55 ml) and water (55 ml),intermediate 8 (23.4 g, 84.28 mmol) and sodium carbonate (13.3 g, 126.42mmol) were added and degassed for 30 min.Tetrakis(triphenylphosphine)palladium(0) (2.4 g, 2.10 mmol) was addedunder nitrogen atmosphere and heated to 80° C. After 12 hours, thereaction mixture was filtered through celite, concentrated and extractedwith ethyl acetate. The organic layer was dried over sodium sulphate andconcentrated under reduced pressure. The crude product was trituratedwith diethyl ether, filtered and dried under vacuum to afford the titlecompound as light brown solid (3.2 g, 26% yield) which is used as suchfor the next step.

(RS)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one

To a solution of intermediate 9 (0.080 g, 0.293 mmol) in DMF (2 ml),potassium carbonate (0.081 g, 0.587 mmol) was added and stirred at RTfor 10 min. To this mixture intermediate 1 (0.215 g, 0.587 mmol) wasadded and stirred for 12 hours. The reaction mixture was diluted withwater and extracted with ethyl acetate. The organic layer was dried oversodium sulphate and concentrated under reduced pressure. The crudeproduct was purified by column chromatography with methanol:dichloromethane to afford the title compound as a pale yellow solid(0.045 g). MP: 175-177° C. ¹H-NMR (δ ppm, DMSO-D₆, 400 MHz): δ 8.20 (s,1H), 7.85 (dd, J 81, 3.0 Hz, 1H), 7.48-7.33 (m, 5H), 7.14 (t, J=8.3 Hz,1H), 7.02 (m, 2H), 6.90 (m, 1H), 6.10 (q, J 7.1 Hz, 1H), 5.42 (s, 2H),4.64 (quintet, J 6.0 Hz, 1H), 1.99 (d, J=7.1 Hz, 3H), 1.42 (d, J=6.1 Hz,6H).

(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one(“S-isomer”)

To a solution of intermediate 9 (0.134 g, 0.494 mmol) in THE (2.0 ml),intermediate 6 (0.150 g, 0.494 mmol) and triphenylphosphine (0.194 g,0.741 mml) were added and stirred at RT for 5 min.Diisopropylazodicarboxylate (0.15 ml, 0.749 mmol) was added heated to45° C. After 2 hours, the reaction mixture was quenched with water andextracted with ethyl acetate. The organic layer was dried over sodiumsulphate and concentrated under reduced pressure. The crude product waspurified by column chromatography with ethyl acetate:petroleum ether toafford the title compound as an off-white solid (0.049 g, 20% yield).MP: 139-142° C. Mass: 571.7 (M⁺). Enantiomeric excess: 89.8% asdetermined by HPLC on a chiralpak AD-H column, enriched in the fasteluting isomer (retention time=10.64 min.).

(R)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-ehromen-4-one

To a solution of intermediate 8 (0.284 g, 0.989 mmol) in THE (5.0 ml),intermediate 4 (0.250 g, 0.824 mmol) and tris(4-methoxy)phenylphosphine(0.435 g, 1.23 mml) were added and stirred at RT for 5 min.Diisopropylazodicarboxylate (0.25 ml, 1.23 mmol) was added stirred atRT. After 12 hours, the reaction mixture was quenched with water andextracted with ethyl acetate. The organic layer was dried over sodiumsulphate and concentrated under reduced pressure. The crude product waspurified by column chromatography with ethyl acetate:petroleum ether toafford the title compound as an off-white solid (0.105 g, 22% yield).MP: 145-148° C. Mass: 571.7 (M⁺). Enantiomeric excess: 95.4% asdetermined by HPLC on a chiralpak AD-H column, enriched in the lateeluting isomer (retention time=14.83 min.).

Biological Evaluation Combination of a Compound of Formula A andAnti-CD20 Antibody Example 1: S-Isomer of a Compound of Formula a andUblituximab Combinations Deplete B Cells from Whole Blood

A flow cytometry assay was used to compare the ability of S-isomer ofcompound of formula A, Ublituximab (Ubx), and combinations thereof todeplete B cells from human whole blood (HWB). In this assay, 50 μl ofHWB sample was treated with either S-isomer of a compound of formula A(1000 nM), UBX (100 μg/ml to 0.1 μg/ml), or UBX in combination withS-isomer of a compound of formula A at 1000 nM and incubated for 24 hrsat 37° C. and 5% CO₂. 20 μl of treated sample was taken in a 1.5 mlcentrifuge tube and labeled with CD45 FITC and CD19 PE or CD20 FITCantibody and incubated in the dark for 1 hour at RT. 1 ml of red bloodcell (RBC) lysing solution was added, and tubes were centrifuged at 3000rpm for 10 minutes. The supernatant was aspirated, and 250 of PBS wasadded to the pellet. The tubes were vortexed, and 5000 events wereacquired on a Guava© easyCyte™ flow cytometer and analyzed with IncyteSoftware.

Gated population of CD45-positive cells were further analyzed for CD19.The number of cells that were positive for CD45 and CD19 was calculated,and the data was expressed as the percentage of CD19 positive cells inthe population. CD20-positive populations were gated with fluorescencepositive minus unlabeled cells, and the data was expressed as thepercentage of CD20-positive cells in the population. The loss ofCD19/CD20 population from control was calculated and expressed as %depletion with respect to control.

The results are shown in FIG. 1. S-isomer of a compound of formula A isnot cytotoxic to B-cells at concentrations up to 10 μM. Therefore, areduction in CD19-positive or CD20-positive HWB B-cells was not observedwith 1 μM S-isomer of a compound of formula A. The UBX anti-CD20antibody resulted in only 20-30% depletion of B-cells at doses from 1 to100,000 ng/ml, but it caused a dose-dependent reduction in CD20+B-cells. Combination of 1000 nM S-isomer of a compound of formula A with10 ng/ml UBX resulted in the potentiation of CD19+ cell depletion, andcombination of 1000 nM S-isomer of a compound of formula A with 0.1-10ng/ml concentrations UBX resulted in a modest additive effect on CD20+cell depletion. These results demonstrate that the combination ofS-isomer of a compound of formula A (1000 nM) with UBX (10 ng/ml)displayed potentiation of CD19-positive cell depletion and a modesteffect on CD20-positive cell depletion.

Example 2: S-Isomer of a Compound of Formula a and UBX CombinationsEffect LPS-Induced B Cell Proliferation

Flow cytometry was used to study the effect of S-isomer of a compound offormula A, Ubx, and combinations thereof on LPS-induced proliferation ofCD19 and CD20 cells in HWB. In these experiments, 250 of diluted (1:3.5with RPMI-HG Media) HWB sample was treated with either S-isomer of acompound of formula A (10 μM to 0.1 μM), UBX (100 μg/ml to 0.1 μg/ml) orUBX with S-isomer of a compound of formula A at 1000 nM for 15 minutesfollowed by 20 μg/ml LPS induction and incubated for 72 hrs at 37° C.and 5% CO₂. 20 μl of treated sample was taken in a 1.5 ml centrifugetube and labeled with CD20 FITC and CD19 PE antibody and incubated inthe dark for 1 hour at RT. 1 ml of RBC lysing solution was added andtubes were centrifuged at 3000 rpm for 10 minutes. Supernatant wasaspirated, and 250 of PBS was added to the pellet. Tubes were vortexed,and 5000 events were acquired on a Guava© easyCyte™ flow cytometer andanalyzed with Incyte Software.

Gated population of lymphocytes positive cells were further analyzed forCD19 and CD20. Cells positive for CD19 and/or CD20 were calculated, anddata were expressed as the percentage positive cells in the population.The loss of positive population from control with LPS induction wascalculated and expressed as % inhibition with respect to control.

The results are shown in FIGS. 2 and 3. S-isomer of a compound offormula A (1 M) caused a dose-dependent inhibition of LPS induced CD19+B-cell proliferation using HWB (˜60%). Addition of 1 M S-isomer of acompound of formula A to different concentrations of UBX did notincrease the response beyond 60% as a result of the minimal effect ofUBX on CD19+ cells. However, an additive effect of the combination onCD19+ cell proliferation was noticed at the 100 ng/ml concentration ofUBX.

In contrast to its effect on CD19+ cell proliferation, S-isomer of acompound of formula A displayed 40% inhibition of CD20+ cells at 1 M. Anadditive effect of the combination of 1 M S-isomer of a compound offormula A with UBX was evident, especially for the 0.1 ng/ml dose.

Example 3: S-Isomer of a Compound Formula a and UBX CombinationsIncrease Apoptosis in Cancer Cells

In order to determine the effect of S-isomer of a compound of formula A,UBX, and combinations thereof on apoptosis in cancer cells, an in situcaspase-3 kit (Millipore) was used. Cells were plated in a 6 well plateat a concentration of 0.5×10⁶ cells/ml, treated with either S-isomer ofa compound of formula A (1000 nM), UBX (100 μg/ml to 0.1 μg/ml), or UBXwith S-isomer of a compound of formula A at 1000 nM, and incubated for24 hrs at 37° C. and 5% CO₂. The cells were then transferred tomicrofuge tubes to receive 10 μl of freshly prepared FLICA™ reagent andincubated for 1 hour at 37° C. and 5% CO₂ away from light. Afterextensive washes with wash buffer, test samples were adjusted toequalize the number of cells in PBS. 100 μl of each cell suspension wastransferred to black 96-well plates in duplicates, and the fluorescencewas read at an excitation wavelength of 490 nm and an emissionwavelength of 520 nm in a plate reader. The fluorescence intensity for aDMSO control was subtracted from that of test compounds. Data wasexpressed as a percent of the maximum response (100%) and plottedaccordingly.

The results are shown in FIG. 4. UBX displayed a limited ability toinduce Caspase-3 activity in the cell lines tested. Caspase-3 activitywas increase by 40-75% by incubating cell lines with 1 M S-isomer of acompound of formula A. A synergistic effect of the combination wasnoticed at 100 ng/ml UBX concentration in Daudi cells while an additiveeffect was seen in RPMI-8226, Raji, and U226B1 cell lines at higherconcentrations (10 & 100 ng/ml) of UBX.

Example 4: S-isomer of a Compound of Formula A and UBX CombinationCauses Cell Cycle Arrest

A cell cycle assay reagent (Millipore) was used to determine the effectof S-isomer of a compound of formula A, UBX, and combinations thereof onthe cell cycle in cancerous cells. In these experiments, cells wereplated in a 6-well plate at a concentration of 0.5×10⁶ cells/ml, treatedwith either S-isomer of a compound of formula A (10 M to 0.1 μM), UBX(100 μg/ml to 0.1 μg/ml), or UBX with S-isomer of a compound of formulaA at 1000 nM, and incubated for 72 hrs at 37° C. and 5% CO₂. The cellswere transferred to microfuge tubes to receive 50 of cell cycle reagentand incubated for 30 minutes at RT away from light. Cells were thendiluted with 300-400 PBS, and a minimum of 10,000 events were acquiredon a Guava© easyCyte™ flow cytometer. The data was analyzed with ExpressPro software and the percentage of the cell population in different cellcycle stages with respect to control was presented in histograms.

FIGS. 5-10 show the results obtained with U266B1, and Raji cells. Inaddition, Tables 1-12 below provide the quantitative results obtainedusing U266B1, DB, Raji, and Daudi cells.

TABLE 1 U266B1 Cells - 72 h Incubation with a Compound of Formula ATreatment G0/G1 S G2/M Sub G0 Control 60.22 4.29 31.72 3.01 10,000 nM2.00 0.94 64.31 28.88 1000 nM 47.80 4.69 47.13 0.88 100 nM 48.69 5.4945.76 0.75 10 nM 55.33 5.38 39.28 0.76 1 nM 57.73 4.87 36.19 0.94 0.1 nM59.26 6.55 30.71 2.75

TABLE 2 U266B1 Cells - 72 h Incubation with UBX Anti-CD20 AntibodyTreatment G0/G1 S G2/M Sub G0 Control 60.22 4.29 31.72 3.01 100 ng/ml52.95 7.91 39.65 1.95 10 ng/ml 56.85 6.34 37.86 0.78 1 ng/ml 58.81 7.0736.07 0.51 0.1 ng/ml 57.53 7.63 35.64 1.35 0.01 ng/ml 59.32 6.57 35.480.74 0.001 ng/ml 60.79 6.00 30.49 1.34

TABLE 3 U266B1 Cells - 72 h Incubation with UBX + a Compound of FormulaA (1000 nM) Treatment G0/G1 S G2/M Sub G0 Control 60.22 4.29 31.72 3.01100 ng/ml + Comp. A 1.25 0.68 84.46 12.76 10 ng/ml + Comp. A 1.68 1.2084.97 11.70 1 ng/ml + Comp. A 47.97 3.81 46.37 0.63 0.1 ng/ml + Comp. A47.31 4.28 46.47 0.50 0.01 ng/ml + Comp. A 47.48 3.84 46.51 0.56 0.001ng/ml + Comp. A 47.09 3.97 47.05 0.59

TABLE 4 DB Cells - 72 h Incubation with a Compound of Formula ATreatment G0/G1 S G2/M Sub G0 Control 54.30 8.00 36.38 0.48 10,000 nM1.40 0.89 94.87 2.06 1000 nM 0.75 0.42 92.79 5.36 100 nM 41.21 6.9650.22 0.43 10 nM 47.22 4.98 46.85 0.55 1 nM 50.61 6.53 41.44 0.54 0.1 nM54.37 4.84 39.41 0.84

TABLE 5 DB Cells - 72 h Incubation with UBX Anti-CD20 Antibody TreatmentG0/G1 S G2/M Sub G0 Control 54.30 8.00 36.38 0.48 100 ng/ml 45.98 7.6646.55 0.67 10 ng/ml 49.52 10.07 40.56 0.56 1 ng/ml 51.36 6.46 40.07 0.520.1 ng/ml 56.34 12.34 38.40 1.10 0.01 ng/ml 54.99 7.73 34.79 0.73 0.001ng/ml 54.38 9.45 34.02 0.59

TABLE 6 DB Cells - 72 h Incubation with UBX + a Compound of Formula ATreatment G0/G1 S G2/M Sub G0 Control 54.30 8.00 36.38 0.48 100 ng/ml +Comp. A 0.48 0.32 93.78 5.91 10 ng/ml + Comp. A 0.70 0.99 93.34 4.79 1ng/ml + Comp. A 0.31 0.74 92.65 6.00 0.1 ng/ml + Comp. A 0.44 0.63 92.845.83 0.01 ng/ml + Comp. A 0.09 0.84 93.97 4.30 0.001 ng/ml + Comp. A0.06 0.17 94.48 5.31

TABLE 7 Raji Cells - 72 h Incubation with a Compound of Formula ATreatment G0/G1 S G2/M Sub G0 Control 54.10 9.08 33.54 2.14 10,000 nM10.12 23.17 58.09 4.90 1000 nM 52.04 3.92 41.21 1.29 100 nM 52.81 6.7237.80 1.04 10 nM 55.96 5.80 34.81 1.06 1 nM 56.93 5.51 34.13 1.89 0.1 nM56.54 8.38 33.63 1.32

TABLE 8 Raji Cells- 72 h Incubation with UBX Anti-CD20 AntibodyTreatment G0/G1 S G2/M Sub G0 Control 54.10 9.08 33.54 2.14 100 ng/ml22.19 11.05 33.74 34.78 10 ng/ml 45.01 8.15 12.90 31.86 1 ng/ml 39.7214.82 15.35 27.32 0.1 ng/ml 41.11 8.93 22.00 23.73 0.01 ng/ml 54.5412.65 25.08 5.51 0.001 ng/ml 50.52 10.61 33.66 4.35

TABLE 9 Raji Cells - 72 h Incubation with UBX + a Compound of Formula A(1000 nM) Treatment G0/G1 S G2/M Sub G0 Control 54.10 9.08 33.54 2.14100 ng/ml + Comp. A 44.19 3.20 0.21 51.17 10 ng/ml + Comp. A 46.93 5.983.20 42.80 1 ng/ml + Comp. A 46.35 6.75 5.98 40.10 0.1 ng/ml + Comp. A44.85 9.72 13.00 30.88 0.01 ng/ml + Comp. A 50.11 12.22 24.04 13.510.001 ng/ml + Comp. A 49.23 5.16 38.21 4.73

TABLE 10 Daudi Cells - 72 h Incubation with a Compound of Formula ATreatment G0/G1 S G2/M Sub G0 Control 50.91 11.10 28.51 10.81 10,000 nM2.53 21.92 65.03 5.01 1000 nM 48.27 7.91 40.10 2.22 100 nM 47.39 11.3338.05 1.30 10 nM 46.84 12.55 37.79 1.68 1 nM 48.11 13.27 36.75 0.74 0.1nM 49.56 14.13 33.23 0.34

TABLE 11 Daudi Cells - 72 h Incubation with UBX Anti-CD20 AntibodyTreatment G0/G1 S G2/M Sub G0 Control 50.91 11.10 28.51 10.81 100 ng/ml43.66 9.28 28.55 19.55 10 ng/ml 40.92 13.23 28.08 17.77 1 ng/ml 40.5217.54 26.99 14.95 0.1 ng/ml 37.40 17.06 32.42 13.42 0.01 ng/ml 36.2419.15 37.83 6.77 0.001 ng/ml 38.12 17.90 41.95 2.03

TABLE 12 Daudi Cells - 72 h Incubation with UBX + a Compound of FormulaA (1000 nM) Treatment G0/G1 S G2/M Sub G0 Control 50.91 11.10 28.5110.81 100 ng/ml + Comp. A 38.37 8.43 12.95 40.24 10 ng/ml + Comp. A49.56 8.47 9.80 32.17 1 ng/ml + Comp. A 51.73 8.75 11.81 26.26 0.1ng/ml + Comp. A 56.23 7.25 11.60 24.80 0.01 ng/ml + Comp. A 55.87 6.1717.00 22.02 0.001 ng/ml + Comp. A 36.58 17.03 35.74 10.64

These results demonstrate that of cells contacted with S-isomer of acompound of formula A, resulted in a dose-dependent G2/M arrest. Inaddition, treatment with UBX for 72 hours caused a modest G2/M arrest inDiffuse Large B-cell Lymphoma (DB) and U266B1 cells, while it increasedthe number of Sub GO cells in Raji and Daudi cells. Combination with 1 MS-isomer of a compound of formula A accentuated the UBX response acrossthe cell-lines tested.

Example 5: S-Isomer of a Compound Formula a and UBX CombinationsSynergistically Increase Apoptosis in Cancer Cells

In order to determine the effect of S-isomer of a compound of formula A,UBX, and combinations thereof on apoptosis in cancer cells, an in situcaspase-3 kit (Millipore) was used. Cells were plated in a 6 well plateat a concentration of 0.5×10⁶ cells/ml, treated with either S-isomer ofa compound of formula A (200-5000 nM), UBX (10,000 ng/ml to 10 ng/ml),or UBX with S-isomer of a compound of formula A (as indicated), andincubated for 24 hrs at 37° C. and 5% CO₂. The cells were thentransferred to microfuge tubes to receive 10 μl of freshly preparedFLICA™ reagent and incubated for 1 hour at 37° C. and 5% CO₂ away fromlight. After extensive washes with wash buffer, test samples wereadjusted to equalize the number of cells in PBS. 100 μl of each cellsuspension was transferred to black 96-well plates in duplicates, andthe fluorescence was read at an excitation wavelength of 490 nm and anemission wavelength of 520 nm in a plate reader. The fluorescenceintensity for a DMSO control was subtracted from that of test compounds.Data was expressed as caspase-3 activity a combination index (C.I.)calculated. C.L's less than one indicate synergism, of one indicateadditive effects and greater than one indicates antagonism.

The results are shown in FIG. 11 and Tables 13-15 for DBCL cell lineLY1. UBX induced caspase-3 activity in LY1. Caspase-3 activity wassynergistically increased in the presence of UBX (all concentrations)and S-isomer of a compound of formula A (1000 nM).

The results are shown in FIG. 12 and Tables 16-18 for the Burkittlymphoma cell line Raji. UBX induced caspase-3 activity in Raji LY1.Caspase-3 activity was synergistically increased in the presence of UBX(all concentrations) and S-isomer of a compound of formula A (200 nM).

TABLE 13 LY1 Cells Incubated with S-isomer S-isomer Concentration (nM)Caspase 3 Activity 200 0.06 1000 0.17 5000 0.61

TABLE 14 LY1 Cells Incubated with UBX UBX Concentration (ng/ml) Caspase3 Activity 10 0.23 100 0.32 1000 0.53 10,000 0.59

TABLE 15 LY1 Cells Incubated with S-isomer and UBX COMBINATION S-isomer(nM) UBX (ng/ml) Caspase 3 Activity C.I. 200 10 0.2715 0.6 200 1000.3404 1.2 200 1000 0.5902 0.2 200 10000 0.6317 0.8 1000 10 0.3811 0.51000 100 0.5523 0.2 1000 1000 0.743 0.1 1000 10000 0.8181 0.1 5000 100.7359 0.5 5000 100 0.8339 0.3 5000 1000 0.999 0.0 5000 10000 0.999 0.0

TABLE 16 Raji Cells Incubated with S-isomer S-isomer Concentration (nM)Caspase 3 Activity 200 0.36 1000 0.56 5000 0.80

TABLE 17 Raji Cells Incubated with UBX UBX Concentration (ng/ml) Caspase3 Activity 10 0.18 100 0.31 1000 0.51 10,000 0.60

TABLE 18 Raji Cells Incubated with S-isomer and UBX COMBINATION S-isomer(nM) UBX (ng/ml) Caspase 3 Activity C.I. 200 10 0.696 0.20 200 100 0.8950.04 200 1000 0.999 0.00 200 10000 0.993 0.00 1000 10 0.805 0.49 1000100 0.939 0.11 1000 1000 0.928 0.13 1000 10000 0.866 0.30 5000 10 0.9010.97 5000 100 0.829 2.03 5000 1000 0.861 1.52 5000 10000 0.844 1.80

Tree additional cell lines, LY10 (DLBCL), Toledo (DLBCL) and Daudi(Burkitt lymphoma) were assessed for the effects of combinations ofS-isomer of a compound of formula A and UBX on caspase 3 activity asdescribed above. The results are shown in Tables 19-21. The combinationsalso demonstrated synergistic activation of caspase 3.

TABLE 19 LY10 Cells Incubated with S-isomer and UBX (DLBCL) S-isomer(nM) UBX (ng/mL) Caspase 3 Activity C.I. 5000 50 0.999 0.021 1000 100.421 0.560 200 2 0.119 0.440

TABLE 20 Toledo Cells Incubated with S-isomer and UBX (DLBCL) S-isomer(nM) UBX (ng/mL) Caspase 3 Activity C.I. 5000 50 0.988 0.336 1000 100.685 0.548 200 2 0.136 0.576

TABLE 21 Daudi Cells Incubated with S-isomer and UBX (Burkitt lymphoma)S-isomer (nM) UBX (ng/mL) Caspase 3 Activity C.I. 5000 50 0.999 0.0041000 10 0.594 0.582 200 2 0.296 0.393

Three Mantle cell lymphoma (MCL) cell lines, Jeko, Mayer and Rec-1, wereassessed for the effects of combinations of S-isomer of a compound offormula A and UBX on caspase 3 activity as described above. The resultsare shown in Tables 22-24. The combinations also demonstratedsynergistic activation of caspase 3, with synergy optimized at higherS-isomer and UBX concentrations.

TABLE 22 Jeko Cells Incubated with S-isomer and UBX S-isomer (nM) UBX(ng/mL) Caspase 3 Activity C.I. 5000 50 0.999 0.000 1000 10 0.655 0.274200 2 0.335 0.506

TABLE 23 Maver Cells Incubated with S-isomer and UBX S-isomer (nM) UBX(ng/mL) Caspase 3 Activity C.I. 5000 50 0.999 0.000 1000 10 0.437 0.603200 2 0.213 0.648

TABLE 24 Rec-1 Cells Incubated with S-isomer and UBX S-isomer (nM) UBX(ng/mL) Caspase 3 Activity C.I. 5000 50 0.999 0.000 1000 10 0.484 0.303200 2 0.403 0.132

Overall, these results show that the S-isomer of a compound of formula Aand UBX potently synergize in the activation of caspase 3, a marker forapoptosis, in B-cell lymphoma models.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance. References cited herein are hereby incorporated herein byreference.

What is claimed is:
 1. A method of treating multiple sclerosis whichcomprises administering to a subject an effective amount of acombination of (i) a compound selected from the group consisting of(RS)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-oneand(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one,or a pharmaceutically acceptable salt, solvate, or prodrug of saidcompound; and (ii) an anti-CD20 antibody or antigen-binding fragmentthereof.
 2. The method of claim 1, wherein said anti-CD20 antibody orfragment thereof is selected from the group consisting of antibodies andfragments that are or bind the same epitope as ublituximab, rituximab,ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, ocaratuzumab,PRO131921 and tositumomab.
 3. The method of claim 1, wherein saidantibody or fragment thereof comprises the VH CDR1, CDR2 and CDR3 regionof sequences SEQ ID NO:1, 2, and 3, and the VL CDR1, CDR2 and CDR3region of sequences SEQ ID NO:6, 7, and
 8. 4. The method of claim 3,wherein said antibody or fragment thereof comprises a VH of SEQ ID NO:4and a VL of SEQ ID NO:9.
 5. The method of claim 2, wherein and saidanti-CD20 antibody is ublituximab or an anti-CD20 antibody that binds tothe same epitope as ublituximab.
 6. The method of claim 5, wherein saidanti-CD20 antibody is ublituximab.
 7. The method of any one of claims1-6, wherein said compound is(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-one.8. The method of claim 1, wherein said compound is thep-toluenesulfonate salt of(S)-2-(1-(4-amino-3-(3-fluoro-4-isopropoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-fluoro-3-(3-fluorophenyl)-4H-chromen-4-oneand said anti-CD20 antibody is ublituximab.
 9. The method of claim 1 or8, wherein said compound and said anti-CD20 antibody or fragment areadministered to the subject sequentially or simultaneously.
 10. Themethod of claim 1 or 8, wherein said compound and said anti-CD20antibody or fragment are contained in the same or separatepharmaceutical compositions.
 11. The method of claim 1, furthercomprising administering to the subject at least one additionaltherapeutic agent.
 12. The method of claim 11, wherein said additionaltherapeutic agent is a steroidal anti-inflammatory drug, a non-steroidalanti-inflammatory drug or an immune-selective anti-inflammatory drug.